Nucleotide sequence encoding the enzyme I-SceI and the uses thereof

ABSTRACT

An isolated DNA encoding the enzyme I-SceI is provided. The DNA sequence can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of application Ser. No. 07/971,160, filedNov. 5, 1992, which is a continuation-in-part of application Ser. No.07/879,689, filed May 5, 1992. The entire disclosures of the priorapplications are relied upon and incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to a nucleotide sequence that encodes therestriction endonuclease I-SceI. This invention also relates to vectorscontaining the nucleotide sequence, cells transformed with the vectors,transgenic animals based on the vectors, and cell lines derived fromcells in the animals. This invention also relates to the use of I-SceIfor mapping eukaryotic genomes and for in vivo site directed geneticrecombination.

The ability to introduce genes into the germ line of mammals is of greatinterest in biology. The propensity of mammalian cells to take upexogenously added DNA and to express genes included in the DNA has beenknown for many years. The results of gene manipulation are inherited bythe offspring of these animals. All cells of these offspring inherit theintroduced gene as part of their genetic make-up. Such animals are saidto be transgenic.

Transgenic mammals have provided a means for studying gene regulationduring embryogenesis and in differentiation, for studying the action ofgenes, and for studying the intricate interaction of cells in the immunesystem. The whole animal is the ultimate assay system for manipulatedgenes, which direct complex biological processes.

Transgenic animals can provide a general assay for functionallydissecting DNA sequences responsible for tissue specific ordevelopmental regulation of a variety of genes. In addition, transgenicanimals provide useful vehicles for expressing recombinant proteins andfor generating precise animal models of human genetic disorders.

For a general discussion of gene cloning and expression in animals andanimal cells, see Old and Primrose, “Principles of Gene Manipulation,”Blackwell Scientific Publications, London (1989), page 255 et seq.

Transgenic lines, which have a predisposition to specific diseases andgenetic disorders, are of great value in the investigation of the eventsleading to these states. It is well known that the efficacy of treatmentof a genetic disorder may be dependent on identification of the genedefect that is the primary cause of the disorder. The discovery ofeffective treatments can be expedited by providing an animal model thatwill lead to the disease or disorder, which will enable the study of theefficacy, safety, and mode of action of treatment protocols, such asgenetic recombination.

One of the key issues in understanding genetic recombination is thenature of the initiation step. Studies of homologous recombination inbacteria and fungi have led to the proposal of two types of initiationmechanisms. In the first model, a single-strand nick initiates strandassimilation and branch migration (Meselson and Radding 1975).Alternatively, a double-strand break may occur, followed by a repairmechanism that uses an uncleaved homologous sequence as a template(Resnick and Martin 1976). This latter model has gained support from thefact that integrative transformation in yeast is dramatically increasedwhen the transforming plasmid is linearized in the region of chromosomalhomology (Orr-Weaver, Szostak and Rothstein 1981) and from the directobservation of a double-strand break during mating type interconversionof yeast (Strathern et al. 1982). Recently, double-strand breaks havealso been characterized during normal yeast meiotic recombination (Sunet al. 1989; Alani, Padmore and Kleckner 1990).

Several double-strand endonuclease activities have been characterized inyeast: HO and intron encoded endonucleases are associated withhomologous recombination functions, while others still have unknowngenetic functions (Endo-SceI, Endo-SceII) (Shibata et al. 1984;Morishima et al. 1990). The HO site-specific endonuclease initiatesmating-type interconversion by making a double-strand break near the YZjunction of MAT (Kostriken et al. 1983). The break is subsequentlyrepaired using the intact HML or HMR sequences and resulting in ectopicgene conversion. The HO recognition site is a degenerate 24 bpnon-symmetrical sequence (Nickoloff, Chen, and Heffron 1986; Nickoloff,Singer and Heffron 1990). This sequence has been used as a“recombinator” in artificial constructs to promote intra- andintermolecular mitotic and meiotic recombination (Nickoloff, Chen andHeffron, 1986; Kolodkin, Klar and Stahl 1986; Ray et al. 1988, Rudin andHaber, 1988; Rudin, Sugarman, and Haber 1989).

The two-site specific endonucleases, I-SceI (Jacquier and Dujon 1985)and I-SceII (Delahodde et al. 1989; Wenzlau et al. 1989), that areresponsible for intron mobility in mitochondria, initiate a geneconversion that resembles the HO-induced conversion (see Dujon 1989 forreview). I-SceI, which is encoded by the optional intron Sc LSU.1 of the21S rRNA gene, initiates a double-strand break at the intron insertionsite (Macreadie et al. 1985; Dujon et al. 1985; Colleaux et al. 1986).The recognition site of I-SceI extends over an 18 bp non-symmetricalsequence (Colleaux et al. 1988). Although the two proteins are notobviously related by their structure (HO is 586 amino acids long whileI-SceI is 235 amino acids long), they both generate 4 bp staggered cutswith 3′OH overhangs within their respective recognition sites. It hasbeen found that a mitochondrial intron-encoded endonuclease, transcribedin the nucleus and translated in the cytoplasm, generates adouble-strand break at a nuclear site. The repair events induced byI-SceI are identical to those initiated by HO.

In summary, there exists a need in the art for reagents and methods forproviding transgenic animal models of human diseases and geneticdisorders. The reagents can be based on the restriction enzyme I-SceIand the gene encoding this enzyme. In particular, there exists a needfor reagents and methods for replacing a natural gene with another genethat is capable of alleviating the disease or genetic disorder.

SUMMARY OF THE INVENTION

Accordingly, this invention aids in fulfilling these, needs in the art.Specifically, this invention relates to an isolated DNA encoding theenzyme I-SceI. The DNA has the following nucleotide sequence:

                                ATG CAT ATG AAA AAC ATC AAA AAA AAC CAGGTA ATG 1672                                M   E   M   K   N   I   K   K   N   Q   V   M  12 2671 AAC CTC GGT CCG AAC TCT AAA CTG CTG AAA GAA TAC AAA TCC CAGCTG ATC GAA CTG AAC 2711   13N   L   G   P   N   S   K   L   L   K   E   Y   K   S   Q   L   I   E   L   N  32 2731 ATC GAA CAG TTC GAA GCA GGT ATC GGT CTG ATC CTG GGT GAT GCTTAC ATC CGT TCT CGT 2790   33I   E   Q   F   E   A   G   I   G   L   I   L   G   D   A   Y   I   R   S   R  52 2791 GAT GAA GGT AAA ACC TAC TGT ATG CAG TTC GAG TGG AAA AAC AAAGCA TAC ATG GAC CAC 2852   53D   E   G   K   T   Y   C   M   Q   F   E   W   K   N   K   A   Y   M   D   E  72 2851 GTA TGT CTG CTG TAC GAT CAG TGG GTA CTG TCC CCG CCG CAC AAAAAA GAA CGT GTT AAC 2913   73V   C   L   L   Y   D   Q   W   V   L   S   P   P   E   K   K   E   R   V   N  92 2911 CAC CTG GGT AAC CTG GTA ATC ACC TGG GGC GCC CAG ACT TTC AAACAC CAA GCT TTC AAC 2973   93H   L   G   N   L   V   I   T   W   G   A   Q   T   F   K   E   Q   A   F   N 112 2971 AAA CTG GCT AAC CTG TTC ATC GTT AAC AAC AAA AAA ACC ATC CCGAAC AAC CTG GTT GAA 3030  113K   L   A   N   L   F   I   V   N   N   K   K   T   I   P   N   N   L   V   E 132 3031 AAC TAC CTG ACC CCG ATG TCT CTG GCA TAC TGG TTC ATG GAT GATGGT GGT AAA TGG GAT 3090  133N   Y   L   T   P   M   S   L   A   Y   W   F   M   D   D   G   G   K   W   D 152 3091 TAC AAC AAA AAC TCT ACC AAC AAA TCG ATC GTA CTG AAC ACC CAGTCT TTC ACT TTC GAA 3153  153Y   N   K   N   S   T   N   K   S   I   V   L   N   T   Q   S   F   T   F   E 172 3151 GAA GTA GAA TAC CTG GTT AAG GGT CTG CGT AAC AAA TTC CAA CTGAAC TGT TAC GTA AAA 3213  173E   V   E   Y   L   V   K   G   L   R   M   K   F   Q   L   N   C   Y   V   K 192 3211 ATC AAC AAA AAC AAA CCG ATC ATC TAC ATC GAT TCT ATG TCT TACCTG ATC TTC TAC AAC 3220  193I   N   K   N   K   P   I   I   Y   I   D   S   M   S   Y   L   I   F   Y   N 212 3271 CTG ATC AAA CCG TAC CTG ATC CCG CAG ATG ATG TAC AAA CTG CCGAAC ACT ATC TCC TCC 3330  213L   I   K   P   Y   L   I   P   Q   M   M   Y   K   L   P   N   T   I   S   S 232 3331 GAA ACT TTC CTG AAA TAA  233 E   T   F   L   K   *

This invention also relates to a DNA sequence comprising a promoteroperatively linked to the DNA sequence of the invention encoding theenzyme I-SceI.

This invention further relates to an isolated RNA complementary to theDNA sequence of the invention encoding the enzyme I-SceI and to theother DNA sequences described herein.

In another embodiment of the invention, a vector is provided. The vectorcomprises a plasmid, bacteriophage, or cosmid vector containing the DNAsequence of the invention encoding the enzyme I-SceI.

In addition, this invention relates to E. coli or eukaryotic cellstransformed with a vector of the invention.

Also, this invention relates to transgenic animals containing the DNAsequence encoding the enzyme I-SceI and cell lines cultured from cellsof the transgenic animals.

In addition, this invention relates to a transgenic organism in which atleast one restriction site for the enzyme I-SceI has been inserted in achromosome of the organism.

Further, this invention relates to a method of genetically mapping aeukaryotic genome using the enzyme I-SceI.

This invention also relates to a method for in vivo site directedrecombination in an organism using the enzyme I-SceI.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be more fully described with reference to thedrawings in which:

FIG. 1 depicts the universal code equivalent of the mitochondrial I-SceIgene.

FIG. 2 depicts the nucleotide sequence of the invention encoding theenzyme I-SceI and the amino acid sequence of the natural I-SceI enzyme.

FIG. 3 depicts the I-SceI recognition sequence and indicates possiblebase mutations in the recognition site and the effect of such mutationson stringency of recognition.

FIG. 4 is the nucleotide sequence and deduced amino acid sequence of aregion of plasmid pSCM525. The nucleotide sequence of the inventionencoding the enzyme I-SceI is enclosed in the box.

FIG. 5 depicts variations around the amino acid sequence of the enzymeI-SceI.

FIG. 6 shows Group I intron encoding endonucleases and relatedendonucleases.

FIG. 7 depicts yeast expression vectors containing the synthetic genefor I-SceI.

FIG. 8 depicts the mammalian expression vector PRSV I-SceI.

FIG. 9 is a restriction map of the plasmid pAF100. (See also YEAST,6:521-534, 1990, which is relied upon and incorporated by referenceherein).

FIGS. 10A and 10B show the nucleotide sequence and restriction sites ofregions of the plasmid pAF100.

FIG. 11 depicts an insertion vector pTSMω, pTKMω, and pTTcω containingthe I-SceI site for E. coli and other bacteria.

FIG. 12 depicts an insertion vector pTYW6 containing the I-SceI site foryeast.

FIG. 13 depicts an insertion vector PMLV LTR SAPLZ containing the I-SceIsite for mammalian cells.

FIG. 14 depicts a set of seven transgenic yeast strains cleaved byI-SceI. Chromosomes from FY1679 (control) and from seven transgenicyeast strains with I-SceI sites inserted at various positions alongchromosome XI were treated with I-SceI. DNA was electrophoresed on 1%agarose. (SeaKem) gel in 0.25×TBE buffer at 130 V and 12° C. on aRotaphor apparatus (Biometra) for 70 hrs using 100 sec to 40 secdecreasing pulse times. (A) DNA was stained with ethidium bromide (0.2μg/ml) and transferred to a Hybond N (Amersham) membrane forhybridization. (B) ³²P labelled cosmid pUKG040 which hybridizes with theshortest fragment of the set was used as a probe. Positions ofchromosome XI and shorter chromosomes are indicated.

FIG. 15 depicts the rationale of the nested chromosomal fragmentationstrategy for genetic mapping. (A) Positions of I-SceI sites are placedon the map, irrespective of the left/right orientation (shorterfragments are arbitrarily placed on the left). Fragment sizes asmeasured from PFGE (FIG. 14A) are indicated in kb (note that the sum ofthe two fragment sizes varies slightly due to the limit of precision ofeach measurement). (B) Hybridization with the probe that hybridizes theshortest fragment of the set determines the orientation of each fragment(see FIG. 14B). Fragments that hybridize with the probe (full lines)have been placed arbitrarily to the left. (C) Transgenic yeast strainshave been ordered with increasing sizes of hybridizing chromosomefragments. (D) Deduced I-SceI map with minimal and maximal size ofintervals indicated in kb (variations in some intervals are due tolimitations of PFGE measurements). (E) Chromosome subfragments are usedas probes to assign each cosmid clone to a given map interval or acrossa given I-SceI site.

FIG. 16 depicts mapping of the I-SceI sites of transgenic yeast strainsby hybridization with left end and right end probes of chromosome XI.Chromosomes from FY1679 (control) and the seven transgenic yeast strainswere digested with I-SceI. Transgenic strains were placed in order asexplained in FIG. 15. Electrophoresis conditions were as in FIG. 14. ³²Plabelled cosmids pUKG040 and pUKG066 were used as left end and right endprobes, respectively.

FIG. 17 depicts mapping of a cosmid collection using the nestedchromosomal fragments as probes. Cosmid DNAs were digested with EcoRIand electrophoresed on 0.9% agarose (SeaKem) gel at 1.5 V/cm for 14 hrs,stained with ethidium bromide and transferred to a Hybond N membrane.Cosmids were placed in order from previous hybridizations to helpvisualize the strategy. Hybridizations were carried out serially onthree identical membranes using left end nested chromosome fragmentspurified on PFGE (see FIG. 16) as probes. A: ethidium bromide staining(ladder is the BRL “1 kb ladder”), B: membrane #1, probe: Left tel toA302 site, C: membrane #1, probe: Left tel to M57 site, D: membrane #2,probe: Left tel to H81 site, E: membrane #2, probe: Left tel to T62site, F: membrane #3, probe: Left tel to G41 site, G: membrane #3,probe: Left tel to D304 site, H: membrane #3, probe: entire chromosomeXI.

FIG. 18 depicts a map of the yeast chromosome XI as determined from thenested chromosomal fragmentation strategy. The chromosome is dividedinto eight intervals (with sizes indicated in kb, see FIG. 15D)separated by seven I-SceI sites (E40, A302 . . . ). Cosmid clonesfalling either within intervals or across a given I-SceI site are listedbelow intervals or below interval boundaries, respectively. Cosmidclones that hybridize with selected genes used as probes are indicatedby letters (a-i). They localize the gene with respect to the I-SceI mapand allow comparison with the genetic map (top).

FIG. 19 depicts diagrams of successful site directed homologousrecombination experiments performed in yeast.

FIG. 20. Experimental design for the detection of HR induced by I-Sce I.a) Maps of the 7.5 kb tk-PhleoLacZ retrovirus (G-MtkPL) and of the 6.0kb PhleoLacZ retrovirus (G-MPL), SA is splice acceptor site. G-MtkPLsequences (from G-MtkPL virus) contains PhleoLacZ fusion gene forpositive selection of infected cells (in phleomycin-containing medium)and tk gene for negative selection (in gancyclovir-containing medium).G-MPL sequences (from G-MPL virus) contains only PhleoLacZ sequences. b)Maps of proviral structures following retroviral integration of G-MtkPLand G-MPL. I-Sce I PhleoLacZ LTR duplicates, placing I-Sce I PhleoLacZsequences in the 5′ LTR. The virus vector (which functions as a promotertrap) is transcribed (arrow) by a flanking cellular promoter, P. c)I-Sce I creates two double strand breaks (DSBs) in host DNA liberatingthe central segment and leaving broken chromosome ends that can pairwith the donor plasmid, pVRneo (d). e) Expected recombinant locusfollowing HR.

FIG. 21. A. Scheme of pG-MPL. SD and SA are splice donor and spliceacceptor sites. The structure of the unspliced 5.8 kb (genomic) andspliced 4.2 kb transcripts is shown below. Heavy bar is ³²Pradiolabelled LacZ probe (P). B. RNA Northern blot analysis of a pG MLPtransformed ψ-2 producer clone using polyadenylated RNA. Note that thegenomic and the spliced mRNA are produced at the same high level.

FIG. 22. A. Introduction of duplicated I-Sce I recognition sites intothe genome of mammalian cells by retrovirus integration. Scheme of G-MPLand G-MtkPL proviruses which illustrates positions of the two LTRs andpertinent restriction sites. The size of Bcl I fragments and of I-SceIfragments are indicated. Heavy bar is ³²P radiolabelled LacZ probe (P).B. Southern blot analysis of cellular DNA from NIH3T3 fibroblasts cellsinfected by G-MtkPL and PCC7-S multipotent cells infected by G-MPL. BclI digests demonstrating LTR mediated PhleoLacZ duplication; I-Sce Idigests demonstrating faithful duplication of I-Sce I sites.

FIG. 23. Verification of recombination by Southern. A.: Expectedfragment sizes in kilobase pairs (kb) of provirus at the recombinantlocus. 1) the parental proviral locus. Heavy bar (P) is ³²Pradioactively labelled probe used for hybridization. 2) a recombinantderived after cleavage at the two I-Sce I sites followed by gap repairusing pVR neo (double-site homologous recombination, DsHR). 3) arecombination event initiated by the cleavage at the I-Sce I sites inthe left LTR (single-site homologous recombination, SsHR). B.: Southernanalysis of DNA from NIH3T3/G-MtkPL clones 1 and 2, PCC7-S/G-MPL clones3 and 4 and transformants derived from cotransfection with pCMV(I-SceI+) and pVRneo (1a, 1b, 2a, 3a, 3b and 4a). Kpn I digestion of theparental DNA generates a 4.2 kb fragment containing LacZ fragment.Recombinants 1a and 3a are examples of DsHR Recombinants 1b, 2a, 3b and4a are examples of SsHR.

FIG. 24. Verification of recombination by Northern blot analyses. A.:Expected structure and sizes (in kb) of RNA from PCC7-S/G-MPL clone 3cells before (top) and after (bottom) I-Sce I induced HR with pVRneo.lHeavy bars P1 and P2 are ³²P radioactively labelled probes. B.: Northernblot analysis of the PCC7-S/G-MPL clone 3 recombinant (total RNA). Lane3 is parental cells, lane 3a recombinant cells. Two first lanes wereprobed with LacZ P1, two last lanes are probed with neo P2. parentalPCC7-S/G-MPL clone 3 cells express a 7.0 kb LacZ RNA as expected oftrapping of a cellular promoter leading to expression of acellular-viral fusion RNA. The recombinant clone does not express thisLacz RNA but expresses a neo RNA of 5.0 kb, corresponding to the sizeexpected for an accurate replacement of PhleoLacZ by neo gene.

FIG. 25. Types of recombination events induced by I-Sce I DSBs, a)Schematic drawing of the structure of the recombination substrate. TheG-MtkPL has provirus two LTRs, each containing an I-Sce I recognitionsite and a PhleoLacZ gene. The LTRs are separated by viral sequencescontaining the tk gene. The phenotype of G-MtkPL containing cells isPhleo^(R), GIs^(s), β-Gal±b) Possible modes of intra-chromosomalrecombination. 1) The I-Sce I endonuclease cuts the I-Sce I site in the5′LTR. The 5′ part of U3 of the 5′LTR can pair and recombine with ithomologous sequence in the 3′LTR (by SSA). 2) The I-Sce I endonucleasecuts the I-Sce I site in the 3′LTR. The 3′ part of U3 of the 3′LTR canpair and recombine with its homologous sequence in the 5′LTR (by SSA).3) The I-Sce I endonuclease cuts I-Sce I sites in the two LTRs. The twofree ends can relegate (by an end-joining mechanism). The resultingrecombination product in each of the three models is a solitary LTR (seeright side). No modification would occur in the cellular sequencesflanking the integration site. c) The I-Sce I endonuclease cuts theI-Sce I sites in the two LTRs. The two free ends can be repaired (by agap repair mechanism) using the homologous chromosome. On the right, theresulting recombination product is the deletion of the proviralintegration locus.

FIG. 26. Southern blot analysis of DNA from NIH3T3/G-MtkPL 1 and 2, andPhleoLacZ⁻ recombinants derived from transfections with pCMV(I-Sce. I+)selected in Gancyclovir containing medium. a) Expected fragment sizes inkilobase pair (kbp) of parental provirus after digestion with Pst Iendonuclease. Pst I digestion of the parental DNA NH3T3/G-MtkPL 1generates two fragments of 10 kbp and of the parental NIH3T3/G-MtkPL 2two fragments of 7 kbp and 9 kbp. b) Southern blot analysis of DNAdigested by Pst I from NIH3T3/G-MtkPL 1, and recombinants derived fromtransfection with pCMV(I-Sce I+) (1.1 to 1.5). c) Southern blot analysisof DNA digested by Pst I from NIH3T3/G-MtkPL 2, and recombinants derivedfrom transfection with pCMV(I-Sce I+) (2.1 to 2.6).

Heavy bar is ³²P radiolabelled LacZ probe (P).

FIG. 27. Southern blot analysis of DNA from NIH3T3/G-MtkPL 1 and 2, andPhleoLacZ⁺ recombinants derived from transfections with pCMV(I-Sce I+)and pCMV(I-Sce I−) and selection in Phleomycin and Gancyclovircontaining medium. a) Expected fragment sizes in kbp of parentalprovirus after digestion with Pst I or Bcl I endonuclease. Pst Idigestion of the parental DNA NIH3T3/G-MtkPL 1 generates two fragmentsof 10 kbp. Bcl I digestion of the parental DNA NIH3T3/G-MtkPL 2generates three fragments of 9.2 kbp, 7.2 kbp and 6.0 kbp. a2) Expectedfragment sizes in kbp of recombinants after digestion with Pst I or BclI endonuclease. Pst I digestion of DNA of the recombinant derived fromNIH3T3/G-MtkPL 1 generates one fragment of 13.6 kbp. Bcl I digestion ofthe DNA of the recombinants derived from NIH3T3/G-MtkPL 2 generates twofragments of 9.2 kbp and 6.0 kbp. b) Southern blot analysis of DNA fromNIH3T3/G-MtkPL 1, and recombinants derived from transfection withpCMV(I-Sce I−) and pCMV(I-Sce I+) (1c, 1d). c) Southern analysis of DNAfrom NIH3T3/G-MtkPL 2, and transformants derived from transfection withpCMV(I-Sce I−) (2a, 2b) and pCMV(I-Sce I+) (2c to 2h).

Heavy bar is ³²P radiolabelled LacZ probe (P).

FIG. 28. FIG. 28 is a diagram illustrating the loss of heterozygosity bythe insertion or presence of an I-Sce I site, expression of the enzymeI-Sce I, cleavage at the site, and repair of the double strand break atthe site with the corresponding chromatid.

FIG. 29. FIG. 29 is a diagram illustrating conditional activation of agene. An I-Sce I site is integrated between tandem repeats, and theenzyme I-Sce I is expressed. The enzyme cleaves the double stranded DNAat the I-Sce I site. The double strand break is repaired by single standannealing, yielding an active gene.

FIG. 30. FIG. 30 is a diagram illustrating one step rearrangement of agene by integration of an I-Sce I site or by use of an I-Sce I sitepresent in the gene. A plasmid having either one I-Sce I site within aninactive gene, or two I-Sce I sites at either end of an active genewithout a promoter, is introduced into the cell. The cell contains aninactive form of the corresponding gene. The enzyme I-Sce I cuts theplasmid at the I-Sce I sites, and recombination between the chromosomeand the plasmid yields an active gene replacing the inactive gene.

FIG. 31. FIG. 31 is a diagram illustrating the duplication of a locus.An I-Sce I site and a distal part of the locus are inserted into thegene by classical gene replacement. The I-Sce I site is cleaved by I-SceI enzyme, and the break is repaired by homologous sequences. Thisresults in duplication of the entire locus.

FIG. 32. FIG. 30 is a diagram illustrating the deletion of a locus. TwoI-Sce I sites are added to flank the locus to be deleted. The I-Sce Ienzyme is expressed, and the sites are cleaved. The two remaining endsrecombine, deleting the locus between the two I-Sce I sites.

FIG. 33. FIG. 33 is a diagram of plasmid pG-MtkΔPAPL showing therestriction sites. The plasmid is constructed by deletion of thepolyadenylation region of the tk gene from the pGMtkPL plasmid.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The genuine mitochondrial gene (ref. 8) cannot be expressed in E. coli,yeast or other organisms due to the peculiarities of the mitochondrialgenetic code. A “universal code equivalent” has been constructed by invitro site-directed mutagenesis. Its sequence is given in FIG. 1. Notethat all non-universal codons (except two CTN) have been replacedtogether with some codons extremely rare in E. coli.

The universal code equivalent has been successfully expressed in E. coliand determines the synthesis of an active enzyme. However, expressionlevels remained low due to the large number of codons that are extremelyrare in E. coli. Expression of the “universal code equivalent” has beendetected in yeast.

To optimize gene expression in heterologous systems, a synthetic genehas been designed to encode a protein with the genuine amino acidsequence of I-SceI using, for each codon, that most frequently used inE. coli. The sequence of the synthetic gene is given in FIG. 2. Thesynthetic gene was constructed in vitro from eight syntheticoligonucleotides with partial overlaps. Oligonucleotides were designedto allow mutual priming for second strand synthesis by Klenow polymerasewhen annealed by pairs. The elongated pairs were then ligated intoplasmids. Appropriately placed restriction sites within the designedsequence allowed final assembly of the synthetic gene by in vitroligation. The synthetic gene has been successfully expressed in both E.coli and yeast.

1. I-SceI Gene Sequence

This invention relates to an isolated DNA sequence encoding the enzymeI-SceI. The enzyme I-SceI is an endonuclease. The properties of theenzyme (ref. 14) are as follows:

-   -   I-SceI is a double-stranded endonuclease that cleaves DNA within        its recognition site. I-SceI generates a 4 bp staggered cut with        3′OH overhangs.    -   Substrate: Acts only on double-stranded DNA. Substrate DNA can        be relaxed or negatively supercoiled.    -   Cations: Enzymatic activity requires Mg⁺ (8 mM is optimum). Mn⁺⁺        can replace Mg⁺⁺ but this reduces the stringency of recognition.    -   Optimum conditions for activity: high pH (9 to 10), temperature        20-40° C., no monovalent cations.    -   Enzyme stability: I-SceI is unstable at room temperature. The        enzyme-substrate complex is more stable than the enzyme alone        (presence of recognition sites stabilizes the enzyme.)

The enzyme I-SceI has a known recognition site. (ref. 14.) Therecognition site of I-SceI is a non-symmetrical sequence that extendsover 18 bp as determined by systematic mutational analysis. The sequencereads: (arrows indicate cuts)

5′ TAGGGATAACAGGGTAAT 3′ 3′ ATCCCTATTGTCCCATTA 5′The recognition site corresponds, in part, to the upstream exon and, inpart, to the downstream exon of the intron plus form of the gene.

The recognition site is partially degenerate: single base substitutionswithin the 18 bp long sequence result in either complete insensitivityor reduced sensitivity to the enzyme, depending upon position and natureof the substitution.

The stringency of recognition has been measured on:

-   -   1—mutants of the site.    -   2—the total yeast genome (Saccharomyces cerevisiae, genome        complexity is 1.4×10⁷ bp). Data are unpublished.

Results are:

-   -   1—Mutants of the site: As shown in FIG. 3, there is a general        shifting of stringency, i.e., mutants severely affected in Mg⁺⁺        become partially affected in Mn⁺⁺, mutants partially affected in        Mg⁺⁺ become unaffected in Mn⁺⁺.    -   2—Yeast: In magnesium conditions, no cleavage is observed in        normal yeast. In the same condition, DNA from transgenic yeasts        is cleaved to completion at the artificially inserted I-SceI        site and no other cleavage site can be detected. If magnesium is        replaced by manganese, five additional cleavage sites are        revealed in the entire yeast genome, none of which is cleaved to        completion. Therefore, in manganese the enzyme reveals an        average of 1 site for ca. 3 millions based pairs (5/1.4×10⁷ bp).    -   Definition of the recognition site: important bases are        indicated in FIG. 3. They correspond to bases for which severely        affected mutants exist. Notice however that:    -   1—All possible mutations at each position have not been        determined; therefore a base that does not correspond to a        severely affected mutant may still be important if another        mutant was examined at this very same position.    -   2—There is no clear-cut limit between a very important base (all        mutants are severely affected) and a moderately important base        (some of the mutants are severely affected). There is a        continuum between excellent substrates and poor substrates for        the enzyme.    -   The expected frequency of natural I-SceI sites in a random DNA        sequence is, therefore, equal to (0.25)⁻¹⁸ or (1.5×10⁻¹¹). In        other words, one should expect one natural site for the        equivalent of ca. 20 human genomes, but the frequency of        degenerate sites is more difficult to predict.    -   I-SceI belongs to a “degenerate” subfamily of the        two-dodecapeptide family. Conserved amino acids of the        dodecapeptide motifs are required for activity. In particular,        the aspartic residues at positions 9 of the two dodecapeptides        cannot be replaced, even with glutamic residues. It is likely        that the dodecapeptides form the catalytic site or part of it.    -   Consistent with the recognition site being non-symmetrical, it        is likely that the endonucleolytic activity of I-SceI requires        two successive recognition steps: binding of the enzyme to the        downstream half of the site (corresponding to the downstream        exon) followed by binding of the enzyme to the upstream half of        the site (corresponding to the upstream exon). The first binding        is strong, the second is weaker, but the two are necessary for        cleavage of, DNA. In vitro, the enzyme can bind the downstream        exon alone as well as the intron-exon junction sequence, but no        cleavage results.

The evolutionarily conserved dodecapeptide motifs of intron-encodedI-SceI are essential for endonuclease activity. It has been proposedthat the role of these motifs is to properly position the acidic aminoacids with respect to the DNA sequence recognition domains of the enzymefor the catalysis of phosphodiester bond hydrolysis (ref. P3).

The nucleotide sequence of the invention, which encodes the naturalI-SceI enzyme is shown in FIG. 2. The nucleotide sequence of the gene ofthe invention was derived by dideoxynucleotide sequencing. The basesequences of the nucleotides are written in the 5′----->3′ direction.Each of the letters shown is a conventional designation for thefollowing nucleotides:

A Adenine G Guanine T Thymine C Cytosine.

It is preferred that the DNA sequence encoding the enzyme I-SceI be in apurified form. For instance, the sequence can be free of humanblood-derived proteins, human serum proteins, viral proteins, nucleotidesequences encoding these proteins, human tissue, human tissuecomponents, or combinations of these substances. In addition, it ispreferred that the DNA sequence of the invention is free of extraneousproteins and lipids, and adventitious microorganisms, such as bacteriaand viruses. The essentially purified and isolated DNA sequence encodingI-SceI is especially useful for preparing expression vectors.

Plasmid pSCM525 is a pUC12 derivative, containing an artificial sequenceencoding the DNA sequence of the invention. The nucleotide sequence anddeduced amino acid sequence of a region of plasmid pSCM525 is shown inFIG. 4. The nucleotide sequence of the invention encoding I-SceI isenclosed in the box. The artificial gene is a BamHI-SalI piece of DNAsequence of 723 base pairs, chemically synthesized and assembled. It isplaced under tac promoter control. The DNA sequence of the artificialgene differs from the natural coding sequence or its universal codeequivalent described in Cell (1986), Vol. 44, pages 521-533. However,the translation product of the artificial gene is identical in sequenceto the genuine omega-endonuclease except for the addition of a Met-Hisat the N-terminus. It will be understood that this modified endonucleaseis within the scope of this invention.

Plasmid pSCM525 can be used to transform any suitable E. coli strain andtransformed cells become ampicillin-resistant. Synthesis of theomega-endonuclease is obtained by addition of I.P.T.G. or an equivalentinducer of the lactose operon system.

A plasmid identified as pSCM525 containing the enzyme I-SceI wasdeposited in E. coli strain TG1 with the Collection Nationale deCultures de Microorganismes (C.N.C.M.) of Institut Pasteur in Paris,France on Nov. 22, 1990, under culture collection deposit Accession No.I-1014. The nucleotide sequence of the invention is thus available fromthis deposit.

The gene of the invention can also be prepared by the formation of3′-----5′ phosphate linkages between nucleoside units using conventionalchemical synthesis techniques. For example, the well-knownphosphodiester, phosphotriester, and phosphite triester techniques, aswell as known modifications of these approaches, can be employed.Deoxyribonucleotides can be prepared with automatic synthesis machines,such as those based on the phosphoramidite approach. Oligo- andpolyribonucleotides can also be obtained with the aid of RNA ligaseusing conventional techniques.

This invention of course includes variants of the DNA sequence of theinvention exhibiting substantially the same properties as the sequenceof the invention. By this it is meant that DNA sequences need not beidentical to the sequence disclosed herein. Variations can beattributable to single or multiple base substitutions, deletions, orinsertions or local mutations involving one or more nucleotides notsubstantially detracting from the properties of the DNA sequence asencoding an enzyme having the cleavage properties of the enzyme I-SceI.

FIG. 5 depicts some of the variations that can be made around the I-SceIamino acid sequence. It has been demonstrated that the followingpositions can be changed without affecting enzyme activity:

positions −1 and −2 are not natural. The two amino acids are added dueto cloning strategies. positions 1 to 10: can be deleted. position 36: Gis tolerated. position 40: M or V are tolerated. position 41: S or N aretolerated. position 43: A is tolerated. position 46: V or N aretolerated. position 91: A is tolerated. positions 123 and 156: L istolerated. position 223: A and S are tolerated.It will be understood that enzymes containing these modifications arewithin the scope of this invention.

Changes to the amino acid sequence in FIG. 5 that have been demonstratedto affect enzyme activity are as follows:

position 19: L to S position 38: I to S or N position 39: G to D or Rposition 40: L to O position 42: L to R position 44: D to E, G or Hposition 45: A to E or D position 46: Y to D position 47: I to R or Nposition 80: L to S position 144: D to E position 145: D to E position146: G to E position 147: G to S

It will also be understood that the present invention is intended toencompass fragments of the DNA sequence of the invention in purifiedform, where the fragments are capable of encoding enzymatically activeI-SceI.

The DNA sequence of the invention coding for the enzyme I-SceI can beamplified in the well known polymerase chain reaction (PCR), which isuseful for amplifying all or specific regions of the gene. See e.g., S.Kwok et al., J. Virol., 61:1690-1694 (1987); U.S. Pat. No. 4,683,202;and U.S. Pat. No. 4,683,195. More particularly, DNA primer pairs ofknown sequence positioned 10-300 base pairs apart that are complementaryto the plus and minus strands of the DNA to be amplified can be preparedby well known techniques for the synthesis of oligonucleotides. One endof each primer can be extended and modified to create restrictionendonuclease sites when the primer is annealed to the DNA. The PCRreaction mixture can contain the DNA, the DNA primer pairs, fourdeoxyribonucleoside triphosphates, MgCl₂, DNA polymerase, andconventional buffers. The DNA can be amplified for a number of cycles.It is generally possible to increase the sensitivity of detection byusing a multiplicity of cycles, each cycle consisting of a short periodof denaturation of the DNA at an elevated temperature, cooling of thereaction mixture, and polymerization with the DNA polymerase. Amplifiedsequences can be detected by the use of a technique termed oligomerrestriction (OR). See, R. K. Saiki et al., Bio/Technology 3:1008-1012(1985).

The enzyme I-SceI is one of a number of endonucleases with similarproperties. Following is a listing of related enzymes and their sources.

Group I intron encoded endonucleases and related enzymes are listedbelow with references. Recognition sites are shown in FIG. 6.

Enzyme Encoded by Ref I-SceI Sc LSU-1 intron this work I-SceII Sc cox1-4intron Sargueil et al., NAR (1990) 18, 5659-5665 I-SceIII Sc cox1-3intron Sargueil et al., MGG (1991) 225, 340-341 I-SceIV Sc cox1-5aintron Seraphin et al. (1992) in press I-CeuI Ce LSU-5 intron Marshall,Lemieux Gene (1991) 104, 241-245 I-CreI Cr LSU-1 intron Rochaix(unpublished) I-PpoI Pp LSU-3 intron Muscarella et al., MCB (1990) 10,3386-3396 I-TevI T4 td-1 intron Chu et al., PNAS (1990) 87, 3574-3578and Bell- Pedersen et al. NAR (1990) 18, 3763-3770. I-TevII T4 sunYintron Bell-Pedersen et al. NAR (1990) 18, 3763-3770. I-TevIII RB3nrdB-1 intron Eddy, Gold, Genes Dev. (1991) 5, 1032-1041 HO HO yeastgene Nickoloff et al., MCB (1990) 10, 1174-1179 Endo SceI RF3 yeastmito. gene Kawasaki et al., JBC (1991) 266, 5342-5347

Putative new enzymes (genetic evidence but no activity as yet) areI-CsmI from cytochrome b intron 1 of Chlamydomonas smithii mitochondria(ref. 15), I-PanI from cytochrome b intron 3 of Podospora anserinamitochondria (Jill Salvo), and probably enzymes encoded by introns Ncnd1·1 and Nc cob·! from Neurospora crassa.

The I-endonucleases can be classified as follows:

Class I: Two dodecapeptide motifs, 4 bp staggered cut with 3′ OHoverhangs, cut internal to recognition site

Subclass “I-SceI” Other subclasses I-SceI I-SceII I-SceIV I-SceIIII-CsmI I-CeuI (only one dodecapeptide motif) I-PanI I-CreI (only onedodecapeptide motif) HO TFP1-408 (HO homolog) Endo SceIClass II: GIY-(N₁₀₋₁₁) YIG motif, 2 bp staggered cut with 3′ OHoverhangs, cut external to recognition site:

I-TevI

Class III: no typical structural motifs, 4 bp staggered cut with 3′ OHoverhangs, cut internal to recognition site:

I-PpoI

Class IV: no typical structural motifs, 2 bp staggered cut with 3′ OHoverhangs, cut external to recognition site:

I-TevII

Class V: no typical structural motifs, 2 bp staggered cut with 5′ OHoverhangs:

I-TevIII.

2. Nucleotide Probes Containing the I-SceI Gene of the Invention

The DNA sequence of the invention coding for the enzyme I-SceI can alsobe used as a probe for the detection of a nucleotide sequence in abiological material, such as tissue or body fluids. The probe can belabeled with an atom or inorganic radical, most commonly using aradionuclide, but also perhaps with a heavy metal. Radioactive labelsinclude ³²P, ³H, ¹⁴C, or the like. Any radioactive label can beemployed, which provides for an adequate signal and has sufficienthalf-life. Other labels include ligands that can serve as a specificbinding member to a labeled antibody, fluorescers, chemiluminescers,enzymes, antibodies which can serve as a specific binding pair memberfor a labeled ligand, and the like. The choice of the label will begoverned by the effect of the label on the rate of hybridization andbinding of the probe to the DNA or RNA. It will be necessary that thelabel provide sufficient sensitivity to detect the amount of DNA or RNAavailable for hybridization.

When the nucleotide sequence of the invention is used as a probe forhybridizing to a gene, the nucleotide sequence is preferably affixed toa water insoluble solid, porous support, such as nitrocellulose paper.Hybridization can be carried out using labeled polynucleotides of theinvention and conventional hybridization reagents. The particularhybridization technique is not essential to the invention.

The amount of labeled probe present in the hybridization solution willvary widely, depending upon the nature of the label, the amount of thelabeled probe which can reasonably bind to the support, and thestringency of the hybridization. Generally, substantial excesses of theprobe over stoichiometric will be employed to enhance the rate ofbinding of the probe to the fixed DNA.

Various degrees of stringency of hybridization can be employed. The moresevere the conditions, the greater the complementarity that is requiredfor hybridization between the probe and the polynucleotide for duplexformation. Severity can be controlled by temperature, probeconcentration, probe length, ionic strength, time, and the like.Conveniently, the stringency of hybridization is varied by changing thepolarity of the reactant solution. Temperatures to be employed can beempirically determined or determined from well known formulas developedfor this purpose.

3. Nucleotide Sequences. Containing the Nucleotide Sequence EncodingI-SceI

This invention also relates to the DNA sequence of the inventionencoding the enzyme I-SceI, wherein the nucleotide sequence is linked toother nucleic acids. The nucleic acid can be obtained from any source,for example, from plasmids, from cloned DNA or RNA, or from natural DNAor RNA from any source, including prokaryotic and eukaryotic organisms.DNA or RNA can be extracted from a biological material, such asbiological fluids or tissue, by a variety of techniques including thosedescribed by Maniatis et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, New York (1982). The nucleic acid willgenerally be obtained from a bacteria, yeast, virus, or a higherorganism, such as a plant or animal. The nucleic acid can be a fractionof a more complex mixture, such as a portion of a gene contained inwhole human DNA or a portion of a nucleic acid sequence of a particularmicroorganism. The nucleic acid can be a fraction of a larger moleculeor the nucleic acid can constitute an entire gene or assembly of genes.The DNA can be in a single-stranded or double-stranded form. If thefragment is in single-stranded form, it can be converted todouble-stranded form using DNA polymerase according to conventionaltechniques.

The DNA sequence of the invention can be linked to a structural gene. Asused herein, the term “structural gene” refers to a DNA sequence thatencodes through its template or messenger mRNA a sequence of amino acidscharacteristic of a specific protein or polypeptide. The nucleotidesequence of the invention can function with an expression controlsequence, that is, a DNA sequence that controls and regulates expressionof the gene when operatively linked to the gene.

4. Vectors Containing the Nucleotide Sequence of the Invention

This invention also relates to cloning and expression vectors containingthe DNA sequence of the invention coding for the enzyme I-SceI.

More particularly, the DNA sequence encoding the enzyme can be ligatedto a vehicle for cloning the sequence. The major steps involved in genecloning comprise procedures for Separating DNA containing the gene ofinterest from prokaryotes or eukaryotes, cutting the resulting DNAfragment and the DNA from a cloning vehicle at specific sites, mixingthe two DNA fragments together, and ligating the fragments to yield arecombinant DNA molecule. The recombinant molecule can then betransferred into a host cell, and the cells allowed to replicate toproduce identical cells containing clones of the original DNA sequence.

The vehicle employed in this invention can be any double-stranded DNAmolecule capable of transporting the nucleotide sequence of theinvention into a host cell and capable of replicating within the cell.More particularly, the vehicle must contain at least one DNA sequencethat can act as the origin of replication in the host cell. In addition,the vehicle must contain two or more sites for insertion of the DNAsequence encoding the gene of the invention. These sites will ordinarilycorrespond to restriction enzyme sites at which cohesive ends can beformed, and which are complementary to the cohesive ends on the promotersequence to be ligated to the vehicle. In general, this invention can becarried out with plasmid, bacteriophage, or cosmid vehicles having thesecharacteristics.

The nucleotide sequence of the invention can have cohesive endscompatible with any combination of sites in the vehicle. Alternatively,the sequence can have one or more blunt ends that can be ligated tocorresponding blunt ends in the cloning sites of the vehicle. Thenucleotide sequence to be ligated can be further processed, if desired,by successive exonuclease deletion, such as with the enzyme Bal 31. Inthe event that the nucleotide sequence of the invention does not containa desired combination of cohesive ends, the sequence can be modified byadding a linker, an adaptor, or homopolymer tailing.

It is preferred that plasmids used for cloning nucleotide sequences ofthe invention carry one or more genes responsible for a usefulcharacteristic, such as a selectable marker, displayed by the host cell.In a preferred strategy, plasmids having genes for resistance to twodifferent drugs are chosen. For example, insertion of the DNA sequenceinto a gene for an antibiotic inactivates the gene and destroys drugresistance. The second drug resistance gene is not affected when cellsare transformed with the recombinants, and colonies containing the geneof interest can be selected by resistance to the second drug andsusceptibility to the first drug. Preferred antibiotic markers are genesimparting chloramphenicol, ampicillin, or tetracycline resistance to thehost cell.

A variety of restriction enzymes can be used to cut the vehicle. Theidentity of the restriction enzyme will generally depend upon theidentity of the ends on the DNA sequence to be ligated and therestriction sites in the vehicle. The restriction enzyme is matched tothe restriction sites in the vehicle, which in turn is matched to theends on the nucleic acid fragment being ligated.

The ligation reaction can be set up using well known techniques andconventional reagents. Ligation is carried out with a DNA ligase thatcatalyzes the formation of phosphodiester bonds between adjacent5′-phosphate and the free 3′-hydroxy groups in DNA duplexes. The DNAligase can be derived from a variety of microorganisms. The preferredDNA ligases are enzymes from E. coli and bacteriophage T4. T4 DNA ligasecan ligate DNA fragments with blunt or sticky ends, such as thosegenerated by restriction enzyme digestion. E. coli DNA ligase can beused to catalyze the formation of phosphodiester bonds between thetermini of duplex DNA molecules containing cohesive ends.

Cloning can be carried out in prokaryotic or eukaryotic cells. The hostfor replicating the cloning vehicle will of course be one that iscompatible with the vehicle and in which the vehicle can replicate. Whena plasmid is employed, the plasmid can be derived from bacteria or someother organism or the plasmid can be synthetically prepared. The plasmidcan replicate independently of the host cell chromosome or anintegrative plasmid (episome) can be employed. The plasmid can make useof the DNA replicative enzymes of the host cell in order to replicate orthe plasmid can carry genes that code for the enzymes required forplasmid replication. A number of different plasmids can be employed inpracticing this invention.

The DNA sequence of the invention encoding the enzyme I-SceI can also beligated to a vehicle to form an expression vector. The vehicle employedin this case is one in which it is possible to express the geneoperatively linked to a promoter in an appropriate host cell. It ispreferable to employ a vehicle known for use in expressing genes in E.coli, yeast, or mammalian cells. These vehicles include, for example,the following E. coli expression vectors:

-   pSCM525, which is an E. coli expression vector derived from pUC12 by    insertion of a tac promoter and the synthetic gene for I-SceI.    Expression is induced by IPTG.-   pGEXω6, which is an E. coli expression vector derived from pGEX in    which the synthetic gene from pSCM252 for I-SceI is fused with the    glutathione S transferase gene, producing a hybrid protein. The    hybrid protein possesses the endonuclease activity.-   pDIC73, which is an E. coli expression vector derived from pET-3C by    insertion of the synthetic gene for I-SceI (NdeI-BamHI fragment of    pSCM525) under T7 promoter control. This vector is used in strain    BL21 (DE3) which expresses the T7 RNA polymerase under IPTG    induction.-   pSCM351, which is an E. coli expression vector derived from pUR291    in which the synthetic gene for I-SceI is fused with the Lac Z gene,    producing a hybrid protein.-   pSCM353, which is an E. coli expression vector derived from pEX1 in    which the synthetic gene for I-SceI is fused with the Cro/Lac Z    gene, producing a hybrid protein.

Examples of yeast expression vectors are:

-   pPEX7, which is a yeast expression vector derived from pRP51-Bam O    (a LEU2d derivative of pLG-SD5) by insertion of the synthetic gene    under the control of the galactose promoter. Expression is induced    by galactose.-   pPEX408, which is a yeast expression vector derived from pLG-SD5 by    insertion of the synthetic gene under the control of the galactose    promoter. Expression is induced by galactose.    Several yeast expression vectors are depicted in FIG. 7.

Typical mammalian expression vectors are:

-   pRSV I-SceI, which is a pRSV derivative in which the synthetic gene    (BamHI-PstI fragment from pSCM525) is under the control of the LTR    promoter of Rous Sarcoma Virus. This expression vector is depicted    in FIG. 8.    Vectors for expression in Chinese Hamster Ovary (CHO) cells can also    be employed.    5. Cells Transformed with Vectors of the Invention

The vectors of the invention can be inserted into host organisms usingconventional techniques. For example, the vectors can be inserted bytransformation, transfection, electroporation, microinjection, or bymeans of liposomes (lipofection).

Cloning can be carried out in prokaryotic or eukaryotic cells. The hostfor replicating the cloning vehicle will of course be one that iscompatible with the vehicle and in which the vehicle can replicate.Cloning is preferably carried out in bacterial or yeast cells, althoughcells of fungal, animal, and plant origin can also be employed. Thepreferred host cells for conducting cloning work are bacterial cells,such as E. coli. The use of E. coli cells is particularly preferredbecause most cloning vehicles, such as bacterial plasmids andbacteriophages, replicate in these cells.

In a preferred embodiment of this invention, an expression vectorcontaining the DNA sequence encoding the nucleotide sequence of theinvention operatively linked to a promoter is inserted into a mammaliancell using conventional techniques.

Application of I-SceI for Large Scale Mapping 1. Occurrence of NaturalSites in Various Genomes

Using the purified I-SceI enzyme, the occurrence of natural ordegenerate sites has been examined on the complete genomes of severalspecies. No natural site was found in Saccharomyces cerevisiae, Bacillusanthracis, Borrelia burgdorferi, Leptospira biflexa and L. interrogans.One degenerate site was found on T7 phage DNA.

2. Insertion of Artificial Sites

Given the absence of natural I-SceI sites, artificial sites can beintroduced by transformation or transfection. Two cases need to bedistinguished: site-directed integration by homologous recombination andrandom integration by non-homologous recombination, transposon movementor retroviral infection. The first is easy in the case of yeast and afew bacterial species, more difficult for higher eucaryotes. The secondis possible in all systems.

3. Insertion Vectors

Two types can be distinguished:

-   -   1—Site specific cassettes that introduce the I-SceI site        together with a selectable marker.        For yeast: all are pAF100 derivatives (Thierry et al. (1990)        YEAST 6:521-534) containing the following marker genes:        pAF101: URA3 (inserted in the HindIII site)        pAF103: Neo^(R) (inserted in BglII site)        pAF104: HIS3 (inserted in BglII site)        pAF105: Kan (inserted in BglII site)        pAF106: Kan^(R) (inserted in BglII site)        pAF107: LYS2 (inserted between HindIII and EcoR V)        A restriction map of the plasmid pAF100 is shown in FIG. 9. The        nucleotide sequence and restriction sites of regions of plasmid        pAF100 are shown in FIGS. 10A and 10B.        Many transgenic yeast strains with the I-SceI site at various        and known places along chromosomes are available.    -   2—Vectors derived from transposable elements or retroviruses.        For E. coli and other bacteria: mini Tn5 derivatives containing        the I-SceI site and        pTSm ωStr^(R)        pTKm ωKan^(R) (See FIG. 11)        pTTc ω Tet^(R)        For yeast: pTyω6 is a pD123 derivative in which the I-SceI site        has been inserted in the LTR of the Ty element.

(FIG. 12) For Mammalian Cells:

pMLV LTR SAPLZ: containing the I-SceI site in the LTR of MLV andPhleo-LacZ (FIG. 13). This vector is first grown in Ψ2 cells (3T3derivative, from R. Mulligan). Two transgenic cell lines with the I-SceIsite at undetermined locations in the genome are available: 1009(pluripotent nerve cells, J. F. Nicolas) and D3 (ES cells able togenerate transgenic animals).

4. The Nested Chromosomal Fragmentation Strategy

The nested chromosomal fragmentation strategy for genetically mapping aeukaryotic genome exploits the unique properties of the restrictionendonuclease I-SceI, such as an 18 bp long recognition site. The absenceof natural I-SceI recognition sites in most eukaryotic genomes is alsoexploited in this mapping strategy.

First, one or more I-SceI recognition sites are artificially inserted atvarious positions in a genome, by homologous recombination usingspecific cassettes containing selectable markers or by random insertion,as discussed supra. The genome of the resulting transgenic strain isthen cleaved completely at the artificially inserted I-SceI site(s) uponincubation with the I-SceI restriction enzyme. The cleavage producesnested chromosomal fragments.

The chromosomal fragments are then purified and separated by pulsedfield gel (PFG) electrophoresis, allowing one to “map” the position ofthe inserted site in the chromosome. If total DNA is cleaved with therestriction enzyme, each artificially introduced I-SceI site provides aunique “molecular milestone” in the genome. Thus, a set of transgenicstrains, each carrying a single I-SceI site, can be created whichdefines physical genomic intervals between the milestones. Consequently,an entire genome, a chromosome or any segment of interest can be mappedusing artificially introduced I-SceI restriction sites.

The nested chromosomal fragments may be transferred to a solid membraneand hybridized to a labelled probe containing DNA complementary to theDNA of the fragments. Based on the hybridization banding patterns thatare observed, the eukaryotic genome may be mapped. The set of transgenicstrains with appropriate “milestones” is used as a reference to map anynew gene or clone by direct hybridization.

Example 1 Application of the Nested Chromosomal Fragmentation Strategyto the Mapping of Yeast Chromosome XI

This strategy has been applied to the mapping of yeast chromosome XI ofSaccharamyces cerevisiae. The I-SceI site was inserted at 7 differentlocations along chromosome XI of the diploid strain FY1679, hencedefining eight physical intervals in that chromosome. Sites wereinserted from a URA3-1-I-SceI cassette by homologous recombination. Twosites were inserted within genetically defined genes, TIF1 and FAS1, theothers were inserted at unknown positions in the chromosome from fivenon-overlapping cosmids of our library, taken at random. Agaroseembedded DNA of each of the seven transgenic strains was then digestedwith I-SceI and analyzed by pulsed field gel electrophoresis (FIG. 14A).The position of the I-SceI site of each transgenic strain in chromosomeXI is first deduced from the fragment sizes without consideration of theleft/right orientation of the fragments. Orientation was determined asfollows. The most telomere proximal I-SceI site from this set of strainsis in the transgenic E40 because the 50 kb fragment is the shortest ofall fragments (FIG. 15A). Therefore, the cosmid clone pUKG040, which wasused to insert the I-SceI site in the transgenic E40, is now used as aprobe against all chromosome fragments (FIG. 14B). As expected, pUKG040lights up the two fragments from strain E40 (50 kb and 630 kb,respectively). The large fragment is close to the entire chromosome XIand shows a weak hybridization signal due to the fact that the insert ofpUKG040, which is 38 kb long, contains less than 4 kb within the largechromosome fragment. Note that the entire chromosome XI remains visibleafter I-SceI digestion, due to the fact that the transgenic strains arediploids in which the I-SceI site is inserted in only one of the twohomologs. Now, the pUKG040 probe hybridizes to only one, fragment of allother transgenic strains allowing unambiguous left/right orientation ofI-SceI sites (See FIG. 15B). No significant cross hybridization betweenthe cosmid vector and the chromosome subfragment containing the I-SceIsite insertion vector is visible. Transgenic strains can now be orderedsuch that I-SceI sites are located at increasing distances from thehybridizing end of the chromosome (FIG. 15C) and the I-SceI map can bededuced (FIG. 15D). Precision of the mapping depends upon PFGEresolution and optimal calibration. Note that actual left/rightorientation of the chromosome with respect to the genetic map is notknown at this step. To help visualize our strategy and to obtain moreprecise measurements of the interval sizes between I-SceI sites betweenI-SceI, a new pulsed field gel electrophoresis with the same transgenicstrains now placed in order was made (FIG. 16). After transfer, thefragments were hybridized successively with cosmids pUKG040 and pUKG066which light up, respectively, all fragments from the opposite ends ofthe chromosome (clone pUKG066 defines the right end of the chromosome asdefined from the genetic map because it contains the SIR1 gene. Aregular stepwise progression of chromosome fragment sizes is observed.Note some cross hybridization between the probe pUKG066 and chromosomeIII, probably due to some repetitive DNA sequences.

All chromosome fragments, taken together, now define physical intervalsas indicated in FIG. 15 d. The I-SceI map obtained has an 80 kb averageresolution.

Example 2 Application of the Nested Chromosomal Fragmentation Strategyto the Mapping of Yeast Artificial Chromosome (YAC) Clones

This strategy can be applied to YAC mapping with two possibilities.

-   -   1—insertion of the I-SceI site within the gene of interest using        homologous recombination in yeast. This permits mapping of that        gene in the YAC insert by I-SceI digestion in vitro. This has        been done and works.    -   2—random integration of I-SceI sites along the YAC insert by        homologous recombination in yeast using highly repetitive        sequences (e.g., B2 in mouse or Alu in human). Transgenic        strains are then used as described in ref. P1 to sort libraries        or map genes.

The procedure has now been extended to YAC containing 450 kb of MouseDNA. To this end, a repeated sequence of mouse DNA (called B2) has beeninserted in a plasmid containing the I-SceI site and a selectable yeastmarker (LYS2). Transformation of the yeast cells containing therecombinant YAC with the plasmid linearized within the B2 sequenceresulted in the integration of the I-SceI site at five differentlocations distributed along the mouse DNA insert. Cleavage at theinserted I-SceI sites using the enzyme has been successful, producingnested fragments that can be purified after electrophoresis. Subsequentsteps of the protocol exactly parallels the procedure described inExample 1.

Example 3 Application of Nested Chromosomal Fragments to the DirectSorting of Cosmid Libraries

The nested, chromosomal fragments can be purified from preparative PFGand used as probes against clones from a chromosome X1 specificsublibrary. This sublibrary is composed of 138 cosmid clones(corresponding to eight times coverage) which have been previouslysorted from our complete yeast genomic libraries by colony hybridizationwith PFG purified chromosome X1. This collection of unordered clones hasbeen sequentially hybridized with chromosome fragments taken in order ofincreasing sizes from the left end of the chromosome. Localization ofeach cosmid clone on the I-SceI map could be unambiguously determinedfrom such hybridizations. To further verify the results and to provide amore precise map, a subset of all cosmid clones, now placed in order,have been digested with EcoRI, electrophoresed and hybridized with thenested series of chromosome fragments in order of increasing sizes fromthe left end of the chromosome. Results are given in FIG. 17.

For a given probe, two cases can be distinguished: cosmid clones inwhich all EcoRI fragments hybridize with the probe and cosmid clones inwhich only some of the EcoRI fragments hybridize (i.e., compare pEKG100to pEKG098 in FIG. 17 b). The first category corresponds to clones inwhich the insert is entirely included in one of the two chromosomefragments, the second to clones in which the insert overlaps an I-SceIsite. Note that, for clones of the pEKG series, the EcoRI fragment of 8kb is entirely composed of vector sequences (pWE15) that do nothybridize with the chromosome fragments. In the case where thechromosome fragment possesses the integration vector, a weak crosshybridization with the cosmid is observed (FIG. 17 e).

Examination of FIG. 17 shows that the cosmid clones can unambiguously beordered with respect to the I-SceI map (FIG. 13E), each clone fallingeither in a defined interval or across an I-SceI site. In addition,clones from the second category allow us to place some EcoRI fragmentson the I-SceI maps, while others remain unordered. The complete set ofchromosome XI-specific cosmid clones, covering altogether eight timesthe equivalent of the chromosome, has been sorted with respect to theI-SceI map, as shown in FIG. 18.

5. Partial Restriction Mapping Using I-SceI

In this embodiment, complete digestion of the DNA at the artificiallyinserted I-SceI site is followed by partial digestion with bacterialrestriction endonucleases of choice. The restriction fragments are thenseparated by electrophoresis and blotted. Indirect end labelling isaccomplished using left or right I-Sce half sites. This technique hasbeen successful with yeast chromosomes and should be applicable withoutdifficulty for YAC.

Partial restriction mapping has been done on yeast DNA and on mammaliancell DNA using the commercial enzyme I-SceI. DNA from cells containingan artificially inserted I-SceI site is first cleaved to completion byI-SceI. The DNA is then treated under partial cleavage conditions withbacterial restriction endonucleases of interest (e.g., BamHI) andelectrophoresed along with size calibration markers. The DNA istransferred to a membrane and hybridized successively using the shortsequences flanking the I-SceI sites on either side (these sequences areknown because they are part of the original insertion vector that wasused to introduce the I-SceI site). Autoradiography (or other equivalentdetection system using non radioactive probes) permit the visualizationof ladders, which directly represent the succession of the bacterialrestriction endonuclease sites from the I-SceI site. The size of eachband of the ladder is used to calculate the physical distance betweenthe successive bacterial restriction endonuclease sites.

Application of I-SceI for In Vivo Site Directed Recombination 1.Expression of I-SceI in Yeast

The synthetic I-SceI gene has been placed under the control of agalactose inducible promoter on multicopy plasmids pPEX7 and pPEX408.Expression is correct and induces effects on site as indicated below. Atransgenic yeast with the I-SceI synthetic gene inserted in a chromosomeunder the control of an inducible promoter can be constructed.

2. Effects of Site Specific Double Strand Breaks in Yeast (Refs. 18 andP4)

Effects on Plasmid-Borne I-SceI Sites:

Intramolecular effects are described in detail in Ref. 18.Intermolecular (plasmid to chromosome) recombination can be predicted.

Effects on Chromosome Integrated I-SceI Sites

In a haploid cell, a single break within a chromosome at an artificialI-SceI site results in cell division arrest followed by death (only afew % of survival). Presence of an intact sequence homologous to the cutsite results in repair and 100% cell survival. In a diploid cell, asingle break within a chromosome at an artificial I-SceI site results inrepair using the chromosome homolog and 100% cell survival. In bothcases, repair of the induced double strand break results in loss ofheterozygosity with deletion of the non homologous sequences flankingthe cut and insertion of the non homologous sequences from the donor DNAmolecule.

3. Application for In Vivo Recombination YACs in Yeast

Construction of a YAC vector with the I-SceI restriction site next tothe cloning site should permit one to induce homologous recombinationwith another YAC if inserts are partially overlapping. This is usefulfor the construction of contigs.

4. Prospects for Other Organisms

Insertion of an I-SceI restriction site has been done for bacteria (E.coli, Yersinia entorocolitica, Y. pestis, Y. pseudotuberculosis), andmouse cells. Cleavage at the artificial I-SceI site in vitro has beensuccessful with DNA from the transgenic mouse cells. Expression ofI-SceI from the synthetic gene in mammalian or plant cells should besuccessful.

The I-SceI site has been introduced in mouse cells and bacterial cellsas follows:

-   -   1—Mouse cells:        -   a—Mouse cells (ψ2) were transfected with the DNA of the            vector pMLV LTR SAPLZ containing the I-SceI site using            standard calcium phosphate transfection technique.        -   b—Transfected cells were selected in DMEM medium containing            phleomycin with 5% fetal calf serum and grown under 12% CO₂,            100% humidity at 37° C. until they form colonies.        -   c—Phleomycin resistant colonies were subcloned once in the            same medium.        -   d—Clone MLOP014, which gave a titer of 10⁵ virus particles            per ml, was chosen. This clone was deposited at C.N.C.M. on            May 5, 1992 under culture collection accession No. I-1207.        -   e—The supernatant of this clone was used to infect other            mouse cells (1009) by spreading 10⁵ virus particles on 10⁵            cells in DMEM medium with 10% fetal calf serum and 5 mg/ml            of “polybrain”. Medium was replaced 6 hours after infection            by the same fresh medium.        -   f—24 hours after infection, phleomycin resistant cell were            selected in the same medium as above.        -   g—phleomycin resistant colonies were subcloned once in the            same medium.        -   h—one clone was picked and analyzed. DNA was purified with            standard procedures and digested with I-SceI under optimal            conditions.    -   2—Bacterial cells:        -   Mini Tn 5 transposons containing the I-SceI recognition site            were constructed in E. coli by standard recombinant DNA            procedures. The mini Tn 5 transposons are carried on a            conjugative plasmid. Bacterial conjugation between E. coli            and Yersinia is used to integrate the mini Tn 5 transposon            in Yersinia. Yersinia cells resistant to Kanamycin,            Streptomycin or tetracycline are selected (vectors pTKM-ω,            pTSM-ω and pTTc-ω, respectively).

Several strategies can be attempted for the site specific insertion of aDNA fragment from a plasmid into a chromosome. This will make itpossible to insert transgenes at predetermined sites without laboriousscreening steps. Strategies are:

-   -   1—Construction of a transgenic cell in which the I-SceI        recognition site is inserted at a unique location in a        chromosome. Cotransformation of the transgenic cell with the        expression vector and a plasmid containing the gene of interest        and a segment homologous to the sequence in which the I-SceI        site is inserted.        -   2—Insertion of the I-SceI recognition site next to or within            the gene of interest carried on a plasmid. Cotransformation            of a normal cell with the expression vector carrying the            synthetic I-SceI gene and the plasmid containing the I-SceI            recognition site.    -   3—Construction of a stable transgenic cell line in which the        I-SceI gene has been integrated in the genome under the control        of an inducible or constitutive cellular promoter.        Transformation of the cell line by a plasmid containing the        I-SceI site next to or within the gene of interest.

Site directed homologous recombination: diagrams of successfulexperiments performed in yeast are given in FIG. 19.

PUBLICATIONS CITED IN APPLICATION

-   1. B. Dujon, Sequence of the intron and flanking exons of the    mitochondrial 21 S rRNA gene of yeast strains having different    alleles at the w and RIB 1 loci. Cell (1980) 20, 185-187.-   2. F. Michel, A. Jacquier and B. Dujon, Comparison of fungal    mitochondrial introns reveals extensive homologies in RNA secondary    structure. Biochimie, 1982, 64, 867-881.-   3. F. Michel and B. Dujon, Conservation of RNA secondary structures    in two intron families including mitochondrial-, chloroplast-, and    nuclear-encoded members. The EMBO Journal, 1983, 2, 33-38.-   4. A. Jacquier and B. Dujon, The intron of the mitochondrial 21S    rRNA gene: distribution in different yeast species and sequence    comparison between Kluyveromyces thermotolerans and Saccharomyces    cerevisiae. Mol. Gen. Gent. (1983) 192, 487-499.-   5. B. Dujon and A. Jacquier, organization of the mitochondrial 21S    rRNA gene in Saccharomyces cerevisiae: mutants of the peptidyl    transferase centre and nature of the omega locus in “Mitochondria    1983”, Editors R. J. Schweyen, K. Wolf, F. Kaudewitz, Walter de    Gruyter et Co., Berlin, N.Y. (1983), 389-403.-   6. A. Jacquier and B. Dujon, An intron encoded protein is active in    a gene conversion process that spreads an intron into a    mitochondrial gene. Cell (1985) 41, 383-394.-   7. B. Dujon, G. Cottarel, L. Colleaux, M. Betermier, A. Jacquier, L.    D'Auriol, F. Galibert, Mechanism of integration of an intron within    a mitochondrial gene: a double strand break and the transposase    function of an intron encoded protein as revealed by in vivo and in    vitro assays. In Achievements and perspectives of Mitochondrial    Research”. Vol. II, Biogenesis, E. Quagliariello et al. Eds.    Elsevier, Amsterdam (1985) pages 215-225.-   8. L. Colleaux, L. D'Auriol, M. Betermier, G. Cottarel, A.    Jacquier, F. Galibert, and B. Dujon, A universal code equivalent of    a yeast mitochondrial intron reading frame is expressed into    Escherichia coli as a specific double strand endonuclease.    Cell (1986) 44, 521-533.-   9. B. Dujon, L. Colleaux, A. Jacquier, F. Michel and C. Monteilhet,    Mitochondrial introns as mobile genetic elements: the role of    intron-encoded proteins. In “Extrachromosomal elements in lower    eucaryotes”, Reed B et al. Eds. (1986) Plenum Pub. Corp. 5-27.-   10. F. Michel and B. Dujon, Genetic Exchanges between Bacteriophage    T4 and Filamentous Fungi? Cell (1986) 46, 323.-   11. L. Colleaux, L. D'Auriol, F. Galibert and B. Dujon, Recognition    and cleavage site of the intron encoded omega transposase. PNAS    (1988), 85, 6022-6026.

12. B. Dujon, Group I introns as mobile genetic elements, facts andmechanistic speculations: A Review. Gene (1989), 82, 91-114.

-   13. B. Dujon, M. Belfort, R. A. Butow, C. Jacq, C. Lemieux, P. S.    Perlman, V. M. Vogt, Mobile introns: definition of terms and    recommended nomenclature. Gene (1989), 82, 115-118.-   14. C. Monteilhet, A. Perrin, A. Thierry, L. Colleaux, B. Dujon,    Purification and Characterization of the in vitro activity of    I-SceI, a novel and highly specific endonuclease encoded by a group    I intron. Nucleic Acid Research (1990), 18, 1407-1413.-   15. L. Colleaux, M-R. Michel-Wolwertz, R. F. Matagne, B. Dujon—The    apocytochrome b gene of Chlamydomonas smithii contains a mobile    intron related to both Saccharomyces and Neurospora introns. Mol.    Gen. Genet. (1990) 223, 288-296.-   16. B. Dujon Des introns autonomes et mobiles. Annales de I'Institut    Pasteur/Actualites (1990) 1. 181-194.-   17. A. Thierry, A. Perrin, J. Boyer, C. Fairhead, B. Dujon, B.    Frey, G. Schmitz. Cleavage of yeast and bacteriophage 17 genomes at    a single site using the rare cutter endonuclease I-Sce. I Nuc. Ac.    Res. (1991) 19, 189-190.-   18. A. Plessis, A. Perrin, J. E. Haber, B. Dujon, Site specific    recombination determined by I-SceI, a mitochondrial intron-encoded    endonuclease expressed in the yeast nucleus. GENETICS (1992) 130,    451-460.

ABSTRACTS

-   A1. A. Jacquier, B. Dujon. Intron recombinational insertion at the    DNA level: Nature of a specific receptor site and direct role of an    intron encoded protein. Cold Spring Harbor Symposium 1984.-   A2. I. Colleaux, L. D'Auriol, M. Demariaux, B. Dujon, F. Galibert,    and A. Jacquier, Construction of a universal code equivalent from a    mitochondrial intron encoded transposase gene using oligonucleotide    directed multiple mutagenesis. Colloque International de DNRS    “oligonucleotids et Genetique Moleculaire” Aussois (Savoie) 8-12    Jan. 1985.-   A3. L. Colleaux, D'Auriol, M. Demariaux, B. Dujon, F. Galibert,    and A. Jacquier, Expression in E. coli of a universal code    equivalent of a yeast mitochondrial intron reading frame involved in    the integration of an intron within a gene Cold Spring Harbor    Meeting on “Molecular Biology of Yeast”, Aug. 13-19, 1985.-   A4. B. Dujon, G. Cottarel, L. Colleaux, M. Demariaux, A.    Jacquier, L. D'Auriol, and F. Galibert, Mechanism of integration of    an intron within a mitochondrial gene: a double strand break and the    “transposase” function of an intron encoded protein as revealed by    in vivo and in vitro assays. International symposium on    “Achievements and Perspectives in Mitochondrial Research”, Selva de    Fasono (Brindisi, Italy) 26 Sep. 1985.-   A5. L. Colleaux, G. Cottarel, M. Betermier, A. Jacquier, B.    Dujon, L. D'auriol, and F. Galibert, Mise en evidence de l'activite    endonuclease double brin d'unc protein codee par un intron    mitochondrial de levure. Forum sur la Biologie Moleculaire de la    levure, Bonbannes, France 2-4 Oct. 1985.-   A6. B. Dujon, L. Colleaux, F. Michel and A. Jacquier, Mitochondrial    introns as mobile genetic elements. In “Extrachromosomal elements in    lower eucaryotes”, Urbana, Ill., 1-5 Jun. 1986.-   A7. L. Colleaux and B. Dujon, Activity of a mitochondrial intron    encoded transposase. Yeast Genetics and Molecular Biology Meeting,    Urbana, Ill. 3-6 Jun. 1986.-   A8. L. Colleaux and B. Dujon, The role of a mitochondrial intron    encoded protein. XIIIth International Conference on Yeast Genetics    and Molecular Biology, Banff, Alberta (Canada) 31 Aug.-5 Sep. 1986.-   A9. L. Colleaux, L. D'Aurio, F. Galibert and B. Dujon, Recognition    and cleavage specificity of an intron encoded transposase. 1987    Meeting on Yeast Genetics and Molecular Biology. San Francisco,    Calif. 16-21 Jun. 1987.-   A10. A. Perrin, C. Monteilhet, L. Colleaux and B. Dujon, Biochemical    activity of an intron encoded transposase of Saccharomyces    cerevisiae. Cold Spring Harbor Meeting on “Molecular Biology of    Mitochondria and chloroplasts” 25-30 Aug. 1987 Cold Spring Harbor,    N.Y.-   A11. B. Dujon, A. Jacquier, L. Colleaux, C. Monteilhet, A. Perrin,    “Les Introns autoepissables et leurs proteins” Colloque “Biologie    Moleculaire de la levure: expression genetique chez Saccharomyces”    organise par la Societe francaise de Microbiologie 18 Jan. 1988    Institut Pasteur, Paris.-   A12. L. Colleaux, L. D'Auriol, C. Monteilhet, F. Galibert and B.    Dujon, Characterization of the biochemical activity of an intron    encoded transposase. 14th International Conference on Yeast Genetics    and Molecular Biology. Espoo, Finland, 7-13 Aug. 1988.-   A13. B. Dujon, A group I intron as a mobile genetic element, Albany    Conference sur “RNA: catalysis, splicing, evolution”, Albany, N.Y.,    22-25 Sep. 1988.-   A14. B. Dujon, L. Colleaux, C. Monteilhet, A. Perrin, L.    D'Auriol, F. Galibert, Group I introns as mobile genetic elements:    the role of intron encoded proteins and the nature of the target    site. 14th Annual EMBO Symposium “Organelle genomes and the nucleus”    Heidelberg, 26-29 Sep. 1988.-   A15. L. Colleaux, R. Matagne, B. Dujon, A new mobile mitochondrial    intron provides evidence for genetic exchange between Neurospora and    Chlamydomonas species. Cold Spring Harbor, May 1989.-   A16. L. Colleaux, M. R. Michel-Wolwertz, R. F. Matagne, B. Dujon,    The apoxytochrome b gene of Chlamydomonas smithii contains a mobile    intron related to both Saccharomyces and Neurospora introns. Fourth    International Conference on Cell and Molecular Biology of    Chlamydomonas. Madison, Wis., April 1990.-   A17. B. Dujon, L. Colleaux, E. Luzi, C. Monteilhet, A. Perrin, A.    Plessis, I. Stroke, A. Thierry, Mobile introns, EMBO Workshop on    “Molecular Mechanisms of transposition and its control, Roscoff    (France) June 1990.-   A18. A. Perrin, C. Monteilhet, A. Thierry, E. Luzi, I. Stroke, L.    Colleaux, B. Dujon. I-SceI, a novel double strand site specific    endonuclease, encoded by a mobile group I intron in Yeast. Workshop    on “RecA and Related Proteins” Sacly, France 17-21 Sep. 1990.-   A19. A. Plessis, A. Perrin, B. Dujon, Site specific recombination    induced by double strand endonucleases, HO and I-SceI in yeast.    Workshop on “RecA and Related Proteins” Saclay, France 17-21 Sep.    1990.-   A20. B. Dujon, The genetic propagation of introns 20th FEBS Meeting,    Budapest, Hungary, August 1990.-   A21. E. Luzi, B. Dujon, Analysis of the intron encoded site specific    endonuclease I-SceI by mutagenesis, Third European Congress on Cell    Biology, Florence, Italy, September 1990.-   A22. B. Dujon, Self splicing introns as contagious genetic elements.    Journees Franco-Beiges de Pont a Mousson. October 1990.-   A23. B. Frey, H. Dubler, G. Schmitz, A. Thierry, A. Perrin, J.    Boyer, C. Fairhead, B. Dujon, Specific cleavage of the yeast genome    at a single site using the rare cutter endonuclease I-SceI Human    Genome, Frankfurt, Germany, November 1990.-   A24. B. Dujon, A. Perrin, I. Stroke, E. Luzi, L. Colleaux, A.    Plessis, A. Thierry, The genetic mobility of group I introns at the    DNA level. Keystone Symposia Meeting on “Molecular Evolution of    Introns and Other RNA elements”, Taos, N. Mex., 2-8 Feb. 1991.-   A25. B. Dujon, J. Boyer, C. Fairhead, A. Perrin, A Thierry,    Cartographie chez la levure. Reunion “Strategies d'etablissement des    cartes geniques” Toulouse 30-31 Mai 1991.-   A26. B. Dujon, A. Thierry, Nested chromosomal fragmentation using    the meganuclease I-SceI: a new method for the rapid mapping of the    yeast genome. Elounda, Crete 15-17 Mail 1991.-   A27. A. Thierry, L. Gaillon, F. Galibert, B. Dujon. The chromosome    XI library: what has been accomplished, what is left. Brugge meeting    22-24 Sep. 1991.-   A28. B. Dujon, A. Thierry, Nested chromosomal fragmentation using    the meganuclease I-SceI: a new method for the rapid physical mapping    of the eukaryotic genomes. Cold Spring Harbor 6-10 May 1992.-   A29. A. Thierry, L. Gaillon, F. Galibert, B. Dujon. Yeast chromosome    XI: construction of a cosmid contig. a high resolution map and    sequencing progress. Cold Spring Harbor 6-10 May 1992.

IN PREPARATION

-   P1. A. Thierry and B. Dujon, Nested Chromosomal Fragmentation Using    the Meganuclease I-SceI: Application to the physical mapping of a    yeast chromosome and the direct sorting of cosmid libraries.    Probably Submission to GENOMICS or EMBO J.-   P2. A. Thierry, L. Colleaux and B. Dujon: Construction and    Expression of a synthetic gene coding for the meganuclease I-SceI.    Possible submission: NAR, EMBO J.-   P3. I. Stroke, V. Pelicic and B. Dujon: The evolutionarily conserved    dodecapeptide motifs of intron-encoded I-SceI are essential for    endonuclease function. Submission to EMBO J.-   P4. C. Fairhead and B. Dujon: Consequences of a double strand break    induced in vivo in yeast at specific artificial sites, using the    meganuclease I-SceI. Possible submission to GENETICS, NATURE.-   P5 A. Perrin, and B. Dujon: Asymetrical recognition by the I-SceI    endonuclease on exon and intron sequences reveals a new step in    intron mobility. Possible submission: NATURE

The entire disclosure of all publications and abstracts cited herein isincorporated by reference herein.

Induction of Homologous Recombination in Mammalian Chromosomes Using theI-Sce I System of Saccharomyces cerevisiae Example 4 Introduction

Homologous recombination (HR) between chromosomal and exogenous DNA isat the basis of methods for introducing genetic changes into the genome(5B, 20B). Parameters of the recombination mechanism have beendetermined by studying plasmid sequences introduced into cells (1B, 4B,10B, 12B) and in in vitro system (8B). HR is inefficient in mammaliancells but is promoted by double-strand breaks in DNA.

So far, it has not been possible to cleave a specific chromosomal targetefficiently, thus limiting our understanding of recombination and itsexploitation. Among endonucleases, the Saccharomyces cerevisiaemitochondrial endonuclease I-Sce I (6B) has characteristics which can beexploited as a tool for cleaving a specific chromosomal target and,therefore, manipulating the chromosome in living organisms. I-Sce Iprotein is an endonuclease responsible for intron homing in mitochondriaof yeast, a non-reciprocal mechanism by which a predetermined sequencebecomes inserted at a predetermined site. It has been established thatendonuclease I-Sce I can catalyze recombination in the nucleus of yeastby initiating a double-strand break (17B). The recognition site ofendonuclease I-Sce I is 18 bp long, therefore, the I-Sce I protein is avery rare cutting restriction endonuclease in genomes (22B). Inaddition, as the I-Sce I protein is not a recombinase, its potential forchromosome engineering is larger than that of systems with target sitesrequirement on both host and donor molecules (9B).

We demonstrate here that the yeast I-Sce I endonuclease can efficientlyinduce double-strand breaks in chromosomal target in mammalian cells andthat the breaks can be repaired using a donor molecule that shareshomology with the regions flanking the break. The enzyme catalyzesrecombination at a high efficiency. This demonstrates that recombinationbetween chromosomal DNA and exogenous DNA can occur in mammalian cellsby the double-strand break repair pathway (21B).

Materials and Methods Plasmid Construction

pG-MPL was obtained in four steps: (I) insertion of the 0.3 kb BglII-Sma I fragment (treated with Klenow enzyme) of the Moloney MurineLeukemia Virus (MoMuLV) env gene (25B) containing SA between the Nhe Iand Xba I sites (treated with Klenow enzyme), in the U3 sequence of the3′LTR of MoMuLV, in an intermediate plasmid. (II) insertion in thismodified LTR with linkers adaptors of the 3.5 kb Nco I-Xho I fragmentcontaining the PhleoLacZ fusion gene (15B) (from pUT65 from Caylalaboratory) at the Xba I site next to SA. (III) insertion of this 3′LTR(containing SA and PhleoLacZ), recovered by Sal I-EcoR I doubledigestion in p5′LTR plasmid (a plasmid containing the 5′LTR to thenucleotide number 563 of MoMuLV (26B) between the Xho I and the EcoR Isites, and (VI) insertion of a synthetic I-Sce I recognition site intothe Nco I site in the 3′LTR between SA and PhleoLacZ).

pG-MtkPl was obtained by the insertion (antisense to the retroviralgenome) of the 1.6 kb tk gene with its promoter with linker adaptatorsat the Pst I site of pG-MPL. pVRneo was obtained in two steps (I)insertion into pSP65 (from Promega) linearized by Pst I-EcoR I doubledigestion of the 4.5 kb Pst I to EcoR I fragment of pG-MPL containingthe 3′LTR with the SA and PhleoLacZ, (II) insertion of the 2.0 kb BglII-BamH I fragment. (treated with Klenow enzyme) containing neoPolyAfrom pRSVneo into the Nco I restriction site (treated with Klenowenzyme) of pSP65 containing part of the 3′LTR of G-MPL (between SA andPhleoLacZ).

pCMV(I-Sce I+) was obtained in two steps: (I) insertion of the 0.73 kbBamH I-Sal I, I-Sce I containing fragment (from pSCM525, A. Thierry,personal gift) into the phCMV1 (F. Meyer, personal gift) plasmid cleavedat the BamH I and the Sal I sites, (II) insertion of a 1.6 kb(nucleotide number 3204 to 1988 in SV40) fragment containing thepolyadenylation signal of SV40 into the Pst I site of phCMV1.

pCMV(I-Sce I−) contains the I-Sce I ORF in reverse orientation in thepCMV(I-Sce I+) plasmid. It has been obtained by inserting the BamH I-PstI I-Sce I ORF fragment (treated with Klenow enzyme) into the phCMV PolyAvector linearized by Nsi I and Sal I double-digestion and treated withKlenow enzyme.

Plasmids pG-MPL, pG-MtkPl, pG-MtkΔPAPL have been described. In additionto the plasmids described above, any kind of plasmid vector can beconstructed containing various promoters, genes, polyA site, I-Sce Isite.

Cell Culture and Selection

3T3, PCC7 S, ψ2 are referenced in (7B) and (13B). Cell selection medium:gancyclovir (14B, 23B) was added into the tissue culture medium at theconcentration of 2 μM. Gancyclovir selection was maintained on cellsduring 6 days. G418 was added into the appropriate medium at aconcentration of 1 mg/ml for PCC7-S and 400 μg/ml for 3T3. The selectionwas maintained during all the cell culture. Phleomycin was used at aconcentration of 10 μg/ml.

Cell Lines

ψ cell line was transfected with plasmids containing a proviralrecombinant vector that contain I-Sce I recognition site: pG-MPL,pG-MtkPL, pG-Mtk_(ΔPA)PL

NIH 3T3 Fibroblastic cell line is infected with:

G-MPL. Multiple (more than 30) clones were recovered. The presence of 1to 14 proviral integrations and the multiplicity of the different pointsof integration were verified by molecular analysis.

G-MtkPL. 4 clones were recovered (3 of them have one normal proviralintegration and 1 of them have a recombination between the two LTR sopresent only one I-Sce I recognition site).

Embryonal carcinoma PCC7-S cell line is infected with:

G-MPL. 14 clones were recovered, normal proviral integration.

Embryonic stem cell line D3 is infected with:

G-MPL. 4 clones were recovered (3 have normal proviral integration, 1has 4 proviral integrations).

“Prepared” Mouse Cells:

Insertion of the retrovirus (proviral integration) induces duplicationof LTR containing the I-Sce I site. The cell is heterozygotic for thesite.

Transfection, Infection, Cell Staining and Nucleic Acids Blot Analysis

These procedures were performed as described in (2B, 3B).

Results

To detect I-Sce I HR we have designed the experimental system shown inFIG. 20. Defective recombinant retroviruses (24B) were constructed withthe I-Sce I recognition site and a PhleoLacZ (15B) fusion gene insertedin their 3′LTR (FIG. 20 a). Retroviral integration results in two I-SceI sites distant of 5.8 kb or 7.2 kb from each other into the cell genome(FIG. 20 b). We hypothesized that I-Sce I-induced double-strand breaks(DSB) at these sites (FIG. 20 c) could initiate HR with a donor plasmid(pVRneo, FIG. 20 d) containing sequences homologous to the flankingregions of the DSBs and that non-homologous sequences, carried by thedonor plasmid, could be copied during this recombination (FIG. 20 e).

Introduction of Duplicated I-Sce I Recognition Sites into the Genome ofMammalian Cells by Retrovirus Integration

More specifically, two proviral sequences were used in these studies.The G-MtkPL proviral sequences (from G-MtkPL virus) contain thePhleoLacZ fusion gene for positive selection of transduced cells (inphleomycine-containing medium) and the tk gene for negative selection(in gancyclovir-containing medium). The G-MPL proviral sequences (fromG-MPL virus) contain only the PhleoLacZ sequences. G-MtkPL and G-MPL aredefective recombinant retroviruses (16B) constructed from anenhancerless Moloney murine leukemia provirus. The virus vectorfunctions as a promoter trap and therefore is activated by flankingcellular promoters.

Virus-producing cell lines were generated by transfecting pGMtkPL orG-MPL into the ψ-2 package cell line (13B). Northern blot analysis ofviral transcripts shows (FIG. 21) that the ψ-2-G-MPL line expresses 4.2and 5.8 kb transcripts that hybridized with LacZ probes. Thesetranscripts probably initiate in the 5′LTR and terminate in the 3′LTR.The 4.5 kb transcript corresponds to the spliced message and the 5.8 kbtranscripts to the unspliced genomic message (FIG. 21.A). This verifiedthe functionality of the 5′LTR and of the splice donor and acceptor inthe virus. Similar results have been obtained with ψ-2G-MtkPL. Virus wasprepared from the culture medium of ψ-2 cell lines.

NIH3T3 fibroblasts and PCC7-S multipotent mouse cell lines (7B) werenext infected by G-MtkPL and G-MPL respectively, and clones wereisolated. Southern blot analysis of the DNA prepared from the clonesdemonstrated LTR-mediated duplication of I-Sce I PhleoLacZ sequences(FIG. 22.a). Bcl I digestion generated the expected 5.8 kb (G-MPL) or7.2 kb (G-MtkPL) fragments. The presence of two additional fragmentscorresponding to Bcl I sites in the flanking chromosomal DNAdemonstrates a single proviral target in each clone isolated. Theirvariable size from clone to clone indicates integration of retrovirusesat distinct loci. That I-Sce I recognition sites have been faithfullyduplicated was shown by I-Sce I digests which generated 5.8 kb (G-MPL)fragments or 7.2 kb (G-MtkPL) (FIG. 22.b)

Induction by I-Sce I of Recombination Leading to DNA Exchange

The phenotype conferred to the NIH3T3 cells by G-MtkPL virus isphleo^(R) β-gal⁺ gls^(S) and to PCC7-S by G-MPL is phleo^(R) β-gal⁺(FIG. 20 b). To allow for direct selection of recombination eventsinduced by I-Sce I we constructed pVRneo donor plasmid. In pVRneo theneo gene is flanked by 300 bp homologous to sequences 5′ to the leftchromosomal break and 2.5 kb homologous to sequences 3′ to the rightbreak (FIG. 20 d). A polyadenylation signal was positioned 3′ to the neogene to interrupt the PhleoLacZ message following recombination. If aninduced recombination between the provirus and the plasmid occurs, theresulting phenotype will be neo^(R) and due to the presence of apolyadenylation signal in the donor plasmid the PhleoLacZ gene shouldnot be expressed, resulting in a phleo^(S) β-gal⁻ phenotype.

With G-MtkPL and G-MtkDPQPL, it is possible to select simultaneously forthe gap by negative selection with the tk gene (with gancyclovir) andfor the exchange of the donor plasmid with positive selection with theneo gene (with geneticine). With G-MPL only the positive selection canbe applied in medium containing geneticine. Therefore, we expected toselect for both the HR and for an integration event of the donor plasmidnear an active endogenous promoter. These two events can bedistinguished as an induced HR results in a neo^(R) β-gal⁻ phenotype anda random integration of the donor plasmid results in a neo^(R) β-gal⁺phenotype.

Two different NIH3T3/G-MtkPL and three different PCC7S/G-MPL clones werethen co-transfected with an expression vector for I-Sce I, pCMV(I-SceI+), and the donor plasmid, pVRneo. Transient expression of I-Sce I mayresult in DSBs at I-Sce I sites, therefore promoting HR with pVRneo. Thecontrol is the cotransfection with a plasmid which does not expressI-Sce I, pCMV(I-Sce I−), and pVRneo.

NIH3T3/G-MtkPL clones were selected either for loss of proviralsequences and acquisition of the neo^(R) phenotype (with gancyclovir andgeneticine) or for neo^(R) phenotype only (Table 1). In the first case,neo^(R)gls^(R) colonies were recovered with a frequency of 10⁻⁴ inexperimental series, and no colonies were recovered in the controlseries. In addition, all neo^(R)gls^(R) colonies were β-gal⁻, consistentwith their resulting from HR at the proviral site. In the second case,neo^(R) colonies were recovered with a frequency of 10⁻³ in experimentalseries, and with a 10 to 100 fold lower frequency in the control series.In addition, 90% of the neo^(R) colonies were found to be β-gal⁻ (inseries with pCMV(I-Sce I+)). This shows that expression of I-Sce Iinduces HR between pVR neo and the proviral site and that site directedHR is ten times more frequent than random integration of pVR neo near acellular promoter, and at least 500 times more frequent than spontaneousHR.

TABLE 1 Induced homologous recombination with I-Sce I Selection G418 +Gls G418 I-Sce I expression + − + − β-gal phenotype + − + − + − + − (A)Cell line NIH 3T3/G-MtkPL Clone 1 0 66 0 0 69 581 93 0 Clone 2 0 120 0 015 742 30 0 PCC7-S/G-MPL Clone 3 54 777 7 0 Clone 4 2 91 1 0 Clone 5 7338 3 0 (B) Molecular event RI 0 8 1 6 DsHR 15 0 19 0 SsHR 0 0 4 0 Del 00 1 0TABLE 1: Effect of I-Sce I mediated double-strand cleavage. A. 10⁶ cellsof NIH3T3/G-MtkPL clones 1 and 2 and 5.10⁶ cells of PCC7-S/G-MPL clones3 to 5 were co-transfected with pVRneo and either pCMV(I-Sce I+) orpCMV(I-Sce I−). Cells were selected in the indicated medium: Geneticin(G418) or geneticin+gancyclovir (G418_Gls). The β-gal expressionphenotype was determined by X-gal histochemical staining. If an inducedrecombination between the provirus and pVRneo occurs, the cells acquirea neo^(R) β-gal⁻ phenotype. B. Molecular analysis of a sample ofrecombinant clones. RI: random integration of pVRneo, parental proviralstructure. DsHR: double site HR. SsHR: single site HR. Del: deletion ofthe provirus (see also FIGS. 20 and 23).

Verification of Recombination by Southern and Northern Blot Analysis

The molecular structure of neo^(R) recombinants has been examined bySouthern blot analysis (FIG. 23 and Table 1). HR at I-Sce I sitespredicts that digestion of recombinant DNA generates a 6.4 kb LacZfragment instead of the 4.2 kb parental fragment. All 15 neo^(R) gls^(R)β-gal⁻ recombinants from NIH3T3 cells exhibited only the 6.4 kb Kpn Ifragment. Therefore, the double selection procedure leads to only theexpected recombinants created by gene replacement (Double SiteHomologous Recombinants, DsHR).

The 25 β-gal⁻ recombinants generated from the single selection fell intofour classes: (a) DsHR induced by I-Sce I as above (19 clones); (b)integration of pVRneo in the left LTR as proven by the presence of a 4.2Kpn I fragment (corresponding to PhleoLacZ in the remaining LTR), inaddition to the 6.4 kb fragment (FIG. 23, Table 1, Single siteHomologous Recombinants, SsHR; 3 independent β-gal⁻ recombinants fromclone 3). These clones correspond to I-Sce I-IHR in left DSB only or(less likely) to double crossing over between LTR and pVRneo; (c) randompVRneo integrations (Table 1, Random Integrations, IR) and simultaneousHR (Table 1, Deletion, Del) (1 β-gal⁻ recombinant); and (d) RandompVRneo integration and simultaneous deletion of provirus (1 β-gal⁻recombinant). We suggest that this fourth class corresponds to repair ofDSBs with the homologous chromosome. As expected, all β-gal⁺recombinants from geneticin selection alone, correspond to random pVRneointegrations, whether they originate from the experimental series (eightclones analyzed) or from the control series (six clones analyzed).

We obtained additional evidence that recombination had occurred at theI-Sce I site of PCC7-S/G-MPL 1 by analyzing the RNAs produced in theparental cells and in the recombinant (FIG. 24). Parental PCC7-S/G-MPL 1cells express a 7.0 kb LacZ RNA indicative of trapping of a cellularpromoter leading to expression of a cellular-viral fusion RNA. Therecombinant clone does not express this LacZ RNA but expresses a neo RNAof 5.0 kb. The size of the neo RNA corresponds to the exact sizeexpected for an accurate exchange of PhleoLacZ by neo gene and uses ofthe same cellular and viral splice site (viral PhleoLacZ RNA in the LTRis 3.7 kb and neo RNA in pVRneo is 1.7 kb).

Discussion

The results presented here demonstrate that double-strand breaks can beinduced by the I-Sce I system of Saccharomyces cerevisiae in mammaliancells, and that the breaks in the target chromosomal sequence inducesite-specific recombination with input plasmidic donor DNA.

To operate in mammalian cells, the system requires endogenous I-Sce Ilike activity to be absent from mammalian cells and I-Sce I protein tobe neutral for mammalian cells. It is unlikely that endogenous I-SceI-like actively operates in mammalian cells as the introduction of I-SceI recognition sites do not appear to lead to rearrangement or mutationin the input DNA sequences. For instance, all NIH3T3 and PCC7-S clonesinfected with a retroviruses containing the I-Sce I restriction sitestably propagated the virus. To test for the toxicity of I-Sce I geneproduct, an I-Sce I expressing plasmid was introduced into the NIH3T3cells line (data not shown). A very high percentage of cotransfer of afunctional I-Sce I gene was found, suggesting no selection against thisgene. Functionality of I-Sce I gene was demonstrated by analysis oftranscription, by immunofluorescence detection of the gene product andbiological function (Choulika et al. in preparation).

We next tested whether the endonuclease would cleave a recognition siteplaced on a chromosome. This was accomplished by placing two I-Sce Irecognition sites separated by 5.8 or 7.2 kb on a chromosome in each LTRof proviral structures and by analyzing the products of a recombinationreaction with a targeting vector in the presence of the I-Sce I geneproduct. Our results indicate that in presence of I-Sce I, the donorvector recombines very efficiently with sequences within the two LTRs toproduce a functional neo gene. This suggests that I-Sce I induced veryefficiently double strand breaks in both I-Sce I sites. In addition, asdouble strand breaks were obtained with at least five distinct proviralinsertions, the ability of I-Sce I protein to digest an I-Sce Irecognition site is not highly dependent on surrounding structures.

The demonstration of the ability of the I-Sce I meganuclease to havebiological function on chromosomal sites in mammalian cell paves theroute for a number of manipulations of the genome in living organisms.In comparison with site-specific recombinases (9B, 18B), the I-Sce Isystem is non-reversible. Site specific recombinases locate not only thesites for cutting the DNA, but also for rejoining by bringing togetherthe two partners. In contrast, the only requirement with the I-Sce Isystem is homology of the donor molecule with the region flanking thebreak induced by I-Sce I protein.

The results indicate for the first time that double strand DNA breaks inchromosomal targets stimulate HR with introduced DNA in mammalian cells.Because we used a combination of double strand breaks (DSB) inchromosomal recipient DNA and super-coiled donor DNA, we explored thestimulation by I-Sce I endonuclease of recombination by the doublestrand break repair pathway (21B). Therefore, the induced break isprobably repaired by a gene conversion event involving the concertedparticipation of both broken ends which, after creation ofsingle-stranded region by 5′ to 3′ exonucleolytic digestion, invade andcopy DNA from the donor copy. However, a number of studies ofrecombination in mammalian cells and in yeast (10B, 11B, 19B) suggestthat there is an alternative pathway of recombination termedsingle-strand annealing (SSA). In the SSA pathway, double-strand breaksare substrates in the action of an exonuclease that exposes homologouscomplementary single-strand DNA on the recipient and donor DNA.Annealing of the complementary strand is then followed by a repairprocess that generates recombinants. The I-Sce I system can be used toevaluate the relative importance of the two pathways.

Example 5

This example describes the use of the I-Sce I meganuclease (involved inintron homing of mitochondria of the yeast Saccharomyces cerevisiae)(6B, 28B) to induce DSB and mediate recombination in mammalian cells.I-Sce I is a very rare-cutting restriction endonuclease, with an 18 bplong recognition site (29B, 22B). In viva, I-Sce I endonuclease caninduce recombination in a modified yeast nucleus by initiating aspecific DBS leading to gap repair by the cell (30B, 17B, 21B).Therefore, this approach can potentially be used as a means ofintroducing specific DSB in chromosomal target DNA with a view tomanipulate chromosomes in living cells. The I-Sce I-mediatedrecombination is superior to recombinase system [11] for chromosomeengineering since the latter requires the presence of target sites onboth host and donor DNA molecules, leading to reaction that isreversible.

The I-Sce I endonuclease expression includes recombination events. Thus,I-Sce I activity can provoke site-directed double strand breaks (DSBs)in a mammalian chromosome. At least two types of events occur in therepair of the DSBs, one leading to intra-chromosomal homologousrecombination and the other to the deletion of the transgene. TheseI-Sce I-mediated recombinations occur at a frequency significantlyhigher than background.

Materials and Methods Plasmid Construction

pG-MtkPL was obtained in five steps: (I) insertion of the 0.3 kbp BglII-Sma I fragment (treated with Klenow enzyme) of the Moloney MurineLeukemia Virus (MoMuLV) env gene (25B) containing a splice acceptor (SA)between the Nhe I and Xba I sites (treated with Klenow enzyme), in theU3 sequence of the 3′LTR of MoMuLV, in an intermediate plasmid. (II)Insertion in this modified LTR of a 3.5 kbp Nco I-Xho I fragmentcontaining the PhleoLacZ fusion gene [13] (from pUT65; Cayla Laboratory,Zone Commerciale du Gros, Toulouse, France) at the Xba I site next toSA. (III) Insertion of this 3′LTR (containing SA and PhleoLacZ),recovered by Sal I-EcoR I double digestion in the p5′LTR plasmid (aplasmid containing the 5′LTR up to the nucleotide no 563 of MoMuLV [12])between the Xho I and the EcoR I site. (IV) Insertion of a syntheticI-Sce I recognition site into the Nco I site in the 3′LTR (between SAand PhleoLacZ), and (V) insertion (antisense to the retroviral genome)of the 1.6 kbp tk gene with its promoter with linker adaptators at thePst I site of pG-MPL.

pCMV(I-Sce I+) was obtained in two steps: (I) insertion of the 0.73 kbpBamH I-Sal I, I-Sce I-containing fragment (from pSCM525, donated by A.Thierry) into the phCMV1 (donated by F. Meyer) plasmid cleaved with BamHI and Sal I, (II) insertion of a 1.6 kbp fragment (nucleotide no 3204 to1988 in SV40) containing the polyadenylation signal of SV40 at the Pst Isite of phCMV1.

pCMV(I-Sce I−) contains the I-Sce I ORF in reverse orientation in thepCMV(I-Sce I+) plasmid. It was obtained by inserting the BamH I-Pst II-Sce I ORF fragment (treated with Klenow enzyme) into the phCMV PolyAvector linearized by Nsi I and Sal I double-digestion and treated withKlenow enzyme.

Cell Culture and Selection

T3 and ψ2 are referenced in (7B) and (13B). Cell selection medium:gancyclovir (14B, 23B) was added into the tissue culture medium at theconcentration of 2 μM. Gancyclovir selection was maintained for 6 days.Phleomycine was used at a concentration of 10 μg/ml. Double selectionswere performed in the same conditions.

Transfection, Infection, Cell Staining and Nucleic Acids Blot Analysis

These protocols were performed as described in (2B, 3B).

Virus-Producing Cell Lines

The virus-producing cell line is generated by transfecting pG-MtkPL intothe ψ-2 packaging cell line. Virus was prepared from the filteredculture medium of transfected ψ-2 cell lines. NIH3T3 fibroblasts wereinfected by G MtkPL, and clones were isolated in a Phleomycin-containingmedium.

Results

To assay for I-Sce I endonuclease activity in mammalian cells, NIH3T3cells containing the G-MtkPL provirus were used. The G-MtkPL provirus(FIG. 25 a) contains the tk gene (in place of the gag, pol and env viralgenes), for negative selection in gancyclovir-containing medium and, inthe two LTRs, an I-Sce I recognition site and the PhleoLacZ fusion gene.The PhleoLacZ gene can be used for positive selection of transducedcells in phleomycine-containing medium.

We hypothesized that the expression of I-Sce I endonuclease in thesecells would induce double-strand breaks (DSB) at the I-Sce I recognitionsites that would be repaired by one of the following mechanisms(illustrated in FIG. 25): a) if the I-Sce I endonuclease induces a cutin only one of the two LTRs (FIG. 1-b 1 and 2), sequences that arehomologous between the two LTRs could pair and recombine leading to anintra-chromosomal homologous recombination (i.e. by single strandannealing (SSA) (12B, 10B) or crossing-over); b) If the I-Sce Iendonuclease induces a cut in each of the two LTRs, the two free endscan religate (end joining mechanism (31B) leading to anintra-chromosomal recombination (FIG. 25-b 3); or alternatively c) thegap created by the two DSBs can be repaired by a gap repair mechanismusing sequences either on the homologous chromosome or on otherchromosomal segments, leading to the loss of the proviral sequences(32B) (FIG. 25-c).

The phenotype conferred to the NIH3T3 cells by the G-MtkPL provirus isPhleo^(R)β-Gal⁺ Gls-^(S). In a first series of experiments, we searchedfor recombination by selecting for the loss of the tk gene.NIH3T3/G-MtkPL 1 and 2 (two independent clones with a different proviralintegration site) were transfected with the I-Sce I expression vectorpCMV(I-Sce I+) or with the control plasmid pCMV(I-Sce−) which does notexpress the I-Sce I endonuclease. The cells were then propagated inGancyclovir-containing medium to select for the loss of tk activity. Theresulting Gls^(R) clones were also assayed for β-galactosidase activityby histochemical staining (with X-gal) (Table 1).

TABLE 1 Number and nature of Gls resistant clones pCMV pCMV I-Sce Iexpression (I-SceI+) (I > SceI−) β-Gal activity + − + − NIH3T3/G-MtkPL 111 154 0 0 NIH3T3/G-MtkPL 2 16 196 2 0TABLE 1: Effect of I-Sce I expression on recombination frequency. 1×10⁶cells of NIH3T3/G-MtkPL 1 and 2×10⁶ cells of NIH3T3/G-MtkPL 1 weretransfected with either pCMV(I-Sce I+) or pCMV (I-Sce I−). Cells werecultivated in medium containing gancyclovir. β-Galactosidase phenotypeof the Gls^(R) clones was determined by X-Gal histochemical staining.

In the control series transfected with pCMV(I-SceI−), Gls^(R) resistantclones were found at a low frequency (2 clones for 3×10⁻⁶ treated cells)and the two were β-Gal⁺. In the experimental series transfected withpCMV(I-SceI+), expression of the I-Sce I gene increased the frequency ofGls^(R) clones 100 fold. These clones were either β-Gal⁻ (93%) or β-Gal⁺(7%). Five β-Gal⁻ clones from the NIH3T3/G-MtkPL 1 and six from theNIH3T3/G-MtkPL 2 were analyzed by Southern blotting using Pst I (FIG.26). In the parental DNA, Pst I endonuclease cuts twice in the tk geneof the provirus (FIG. 26 a). The sizes of the two PhleoLacZ containingfragments are determined by the position of the Pst I sites in theflanking cellular DNA. In NIH3T3/G-MtkPL 1, these two PhleoLacZfragments are 10 kbp long and in NIH3T3/G-MtkPL 2 they are 7 and 9 kbplong. The five Gls^(R) β-Gal⁻ resistant clones from NIH3T3/G-MtkPL 1 andthe six clones from th NIH3T3/G-MtkPL 2 all showed an absence of the tkgene and of the two PhleoLacZ sequences (FIGS. 26 b and c).

In the experimental series the number of Gls^(R) β-Gal⁺ clones isincreased about 10 fold by I-Sce I expression in comparison to thecontrol series. These were not analyzed further.

In order to increase the number of Gls^(R) β-Gal⁺ clones recovered, in asecond set of experiments, the cells were grown in a medium containingboth Gancyclovir and Phleomycin. Gancyclovir selects for cells that havelost tk activity and Phleomycin for cells that maintained the PhleoLacZgene. We transfected NIH3T3/G-MtkPLs 1 and 2 with pCMV(I-SceI+) orpCMV(I-SceI−) (Table 2).

TABLE 2 Number of Phleo and Gls resistant clones I-Sce I expression pCMV(I-SceI+) pCMV (I-SceI−) NIH3T3/G-MtkPL 1 74 2 NIH3T3/G-MtkPL 2 207 9TABLE 2: Effect of I-Sce I expression on the intra-chromosomalrecombination frequency. 2×10⁶ cells of NIH3T3/G-MtkPL 1 and 9×10⁶ cellsof NIH3T3/G-MtkPL 2 were transfected with either pCMV(I-Sce I+) orpCMV(I-Sce I−). Cells were cultured in Phleomycin and gancyclovircontaining medium.

In the control series, the frequency of recovery of Phleo^(R) Gls^(R)resistant clones was 1×10⁶. This result reflects cells that havespontaneously lost tk activity, while still maintaining the PhleoLacZgene active. In the experimental series, this frequency was raised about20 to 30 fold, in agreement with the first set of experiments (Table 1).

The molecular structure of the Phleo^(R)β-Gal⁺Gls^(R) clones wasanalyzed by Southern blotting (FIG. 27). Four clones from NIH3T3.G-MtkPL I were analyzed, two from the experimental series and two fromthe control. Their DNA was digested with Pst I endonuclease. If anintra-chromosomal event had occurred, we expected a single Pst Ifragment of 13.6 kbp (that is the sum of the three Pst I fragments ofthe parental DNA minus the I-Sce I fragment, see FIG. 27 a). All fourPhleo^(R)Gls^(R) resistant clones exhibited this 13.6 kbp Pst Ifragment, suggesting a faithful intra-molecular recombination (FIG. 27b).

DNA from eight clones from NIH3T3/G-MtkPL 2 cells were analyzed bySouthern blotting using Bcl I digestion (six from the experimentalseries and two from the control). Bcl I digestion of the parental DNAresults in one 7.2 kbp fragment containing the proviral sequences and intwo flanking fragments of 6 kbp and 9.2 kbp. An intra-chromosomalrecombination should result in the loss of the 7.2 kbp fragment leavingthe two other bands of 6 kbp and 9.2 kbp unchanged (FIG. 27 a). Theeight clones (2.7 to 2.16) showed the disappearance of the tk containing7.2 kbp fragment indicative of an intra-chromosomal recombinationbetween the two LTRs (FIG. 27 c).

Discussion

The results presented here demonstrate that the yeast I-Sce Iendonuclease induces chromosomal recombination in mammalian cells. Thisstrongly suggests that I-Sce I is able to cut in vivo a chromosome at apredetermined target.

Double-strand breaks in genomic sequences of various species stimulaterecombination (21B, 19B). In the diploid yeast, a chromosomal DSB canlead to the use of the homo-allelic locus as a repair matrix. Thisresults in a gene conversion event, the locus then becoming homozygous(30B). The chromosomal DSBs can also be repaired by using homologoussequences of an ectopic locus as matrix (32B). This result is observedat a significant level as a consequence of a DSB gap repair mechanism.If the DSB occurs between two direct-repeated chromosomal sequences, themechanism of recombination uses the single strand annealing (SSA)pathway (11B, 10B). The SSA pathway involves three steps: 1) anexonucleolysis initiated at the point of the break leaving 3′ protrudingsingle-strand DNAs; 2) a pairing of the two single strand DNAs by theirhomologous sequences, 3) a repair of the DNA by repairs complexes andmutator genes which resolve the non-homologous sequences (33B). Aspecial case concerns the haploid yeast for which it has been showedthat DSBs induced by HO or I-Sce I endonucleases in a chromosome leadsto the repair of the break by end joining (34B). This occurs, but at alow efficiency (30B, 35B).

Our results show that the presence of two I-Sce I sites in a proviraltarget and the expression of the I-Sce I endonuclease lead to anincrease in the deletion of a thymidine kinase gene at a frequency atleast 100 fold greater than that occurring spontaneously. Two types oftk deleted clones arise from I-Sce I mediated recombination: clones thathave kept (7%) and clones that have lost (93%) the PhleoLacZ sequences.

The generation of tk⁻PhleoLacZ⁺ cells is probably the consequence ofintra-chromosomal recombination. Studies have shown that in arecombinant provirus with an I-Sce I recognition site in the LTRs, theI-Sce I endonuclease leads in 20% of the cases to the cleavage of onlyone proviral I-Sce I site and in 80% to the cleavage of the two proviralI-Sce I sites. If only one of the two I-Sce I sites is cut by theendonuclease, an intra-chromosomal recombination can occur by the SSApathway. If the two I-Sce I sites are cut, the tk⁻PhleoLacC⁺ cells canbe generated by end joining, allowing intra-chromosomal recombination(see FIG. 1). Although, in the diploid yeast, this pathway is notfavorable (the break is repaired using homologous chromosomal sequences)(2B), it remains possible that this pathway is used in mammalian cells.

The generation of tk⁻/PhleoLacZ⁻ cells is probably a consequence ofeither a homo-allelic and/or an ectopic gene conversion event (36B).Isolation and detailed molecular analysis of the proviral integrationsites will provide information on the relative frequency of each ofthese events for the resolution of chromosomal DSBs by the cell. Thisquantitative information is important as, in mammalian cells, the highredundancy of genomic sequences raises the possibility of a repair ofDSBs by ectopic homologous sequences. Ectopic recombination for repairof DSBs may be involved in genome shaping and diversity in evolution[29].

The ability to digest specifically a chromosome at a predeterminedgenomic location has several potential applications for genomemanipulation.

The protocol of gene replacement described herein can be varied asfollows:

Variety of Donor Vectors

Size and sequence of flanking regions of I-Sce-I site in the donorplasmid (done with 300 pb left and 2.5 kb right): Differentconstructions exist with various size of flanking regions up to a totalof 11 kb left and right from I-Sce I site. The sequences depend from theconstruction (LTR, gene). Any sequence comprising between 3 00 bp to 11kb can be used.

-   -   Inserts (neo, phleo, phleo-LacZ and Pytk-neo have been        constructed). Antibiotic resistance: neomycin, phleomycin;        reporter gene (LacZ); HSV1 thymidine kinase gene: sensitivity to        gancyclovir. It is impossible to insert any kind of gene        sequence up to 10 kb or to replace it. The gene can be expressed        under an inducible or constitutive promoter of the retrovirus,        or by gene trap and homologous recombination (i.e. Insulin, Hbs,        ILs and various proteins).

Various methods can be used to express the enzyme I-Sce I: transienttransfection (plasmid) or direct injection of protein (in embryonucleus); stable transfection (various promoters like: CMV, RSV andMoMuLV); defective recombinant retroviruses (integration of ORF inchromosome under MoMuLV promoter); and episomes.

Variation of host range to integrate I-Sce I site:

Recombinant retroviruses carrying I-Sce I site (i.e. pG-MPL, pG-MtkPL,pG-Mtk_(Δ)PAPL) may be produced in various packaging cell lines(amphotropic or xenotropic).

Construction of Stable Cell Lines Expressing I-Sce I and Cell ProtectionAgainst Retroviral Infection

Stable cell line expressing I-Sce I are protected against infection by aretroviral vector containing I-Sce I site (i.e. NI 3T3 cell lineproducing I-Sce I endonuclease under the control of the CMV promoter isresistant to infection by a pG-MPL or pGMtkPL or I-Sce I under MoMuLVpromoter in ψ2 cells).

Construction of Cell Lines and Transgenic Animals Containing the I-Sce ISite

Insertion of the I-Sce I site is carried out by a classical genereplacement at the desired locus and at the appropriate position. It isthen possible to screen the expression of different genes at the samelocation in the cell (insertion of the donor gene at the artificiallyinserted I-Sce I site) or in a transgenic animal. The effect of multipledrugs, ligands, medical protein, etc., can be tested in a tissuespecific manner. The gene will consistently be inserted at the samelocation in the chromosome.

For “Unprepared” mouse cells, and all eucaryotic cells, a one step genereplacement/integration procedure is carried out as follows:

-   -   Vectors (various donor plasmids) with I-Sce I site: one site        within the gene (or flanking) or two sites flanking the donor        gene.    -   Method to express the enzyme        Transient expression: ORF on the same plasmid or another        (cotransfection).

Specific details regarding the methods used are described above. Thefollowing additional details allow the construction of the following:

a cell line able to produce high titer of a variety of infectiveretroviral particles;

plasmid containing a defective retrovirus with I-Sce I sites,reporter-selector gene, active LTRs and other essential retroviralsequences; a plasmid containing sequences homologous to flanking regionsof I-Sce I sites in above engineered retrovirus and containing amultiple cloning site; and a vector allowing expression of I-Sce Iendonuclease and adapted to the specific applications.

Mouse fibroblast ψ2 cell line was used to produce ectopic defectiverecombinant retroviral vectors containing I-Sce I sites. Cell linesproducing plasmids as pG-MPL, pG-MtkPL, PG-Mtk_(ΔPA)PL are alsoavailable. In addition, any cells, like mouse amphotropic cells lines(such as PA12) or xenotropic cells lines, that produce high titerinfectious particles can be used for the production of recombinantretroviruses carrying I-Sce I site (i.e., pG-MPL, pG-MtkPL,pG-Mtk_(ΔPA)PL) in various packaging cell lines (amphotropic, ectropicor xenotropic).

A variety of plasmids containing I-Sce I can be used in retroviralconstruction, including pG-MPL, pG-MtkPL, and pG-Mtk_(ΔPA)PL. Otherskind of plasmid vector can be constructed containing various promoters,genes, polyA site, and I-Sce I site. A variety of plasmid containingsequences homologs to flanking regions of I-Sce I can be constructed.The size and sequence of flanking regions of I-Sce I site in the donorplasmid are prepared such that 300 kb are to the left and 2.5 kb are tothe right). Other constructions can be used with various sizes offlanking regions of up to about 11 kb to the left and right of the I-SceI recognition site.

Inserts containing neomycin, phleomycin and phleo-LacZ have beenconstructed. Other sequences can be inserted such as drug resistance orreporter genes, including LacZ, HSV1 or thymidine kinase gene(sensibility to gancyclovir), insulin, CFTR, IL2 and various proteins.It is normally possible to insert any kind of sequence up to 12 kb,wherein the size depends on the virus capacity of encapsidation). Thegene can be expressed under inducible or constitutive promoter of theretrovirus, or by gene trap after homologous recombination.

A variety of plasmids containing I-Sce I producing the endonuclease canbe constructed. Expression vectors such as pCMVI-SceI(+) or similarconstructs containing the ORF, can be introduced in cells by transienttransfection, electroporation or lipofection. The protein can also beintroduced directly into the cell by injection of liposomes.

Variety of cells lines with integrated I-Sce I sites can be produced.Preferably, insertion of the retrovirus (proviral integration) induceduplication of LTR containing the I-Sce I site. The cell will behemizygote for the site. Appropriate cell lines include:

1. Mouse Fibroblastic cell line, NIH 3T3 with 1 to 14 proviralintegration of G-MPL. Multiple (more than 30) clones were recovered. Thepresence of and the multiplicity of the different genomic integrations(uncharacterized) were verified by molecular analysis.

2. Mouse Fibroblastic cell line, NIH 3T3 with 1 copy of G-MtkPLintegrated in the genome. 4 clones were covered.

3. Mouse Embryonal Carcinoma cell line, PCC7-S with 1 to 4 copies ofG-MPL proviral integration in the genome. 14 clones were covered.

4. Mouse Embryonal Carcinoma cell line, PCC4 with 1 copy of G-MtkPLintegrated in the genome.

5. Mouse Embryonic Stem cell line D3 with 1 to 4 copies of G-MPL at avariety of genomic localisation (uncharacterized). 4 clones wererecovered.

Construction of other cell lines and transgenic animals containing theI-Sce I site can be done by insertion of the I-Sce I site by a classicalgene replacement at the desired locus and at the appropriate position.Any kind of animal or plant cell lines could a priori be used tointegrate I-Sce I sites at a variety of genomic localisation with celllines adapted. The invention can be used as follows:

1. Site Specific Gene Insertion

The methods allow the production of an unlimited number of cell lines inwhich various genes or mutants of a given gene can be inserted at thepredetermined location defined by the previous integration of the I-SceI site. Such cell lines are thus useful for screening procedures, forphenotypes, ligands, drugs and for reproducible expression at a veryhigh level of recombinant retroviral vectors if the cell line is atranscomplementing cell line for retrovirus production.

Above mouse cells or equivalents from other vertebrates, including man,can be used. Any plant cells that can be maintained in culture can alsobe used independently of whether they have ability to regenerate or not,or whether or not they have given rise to fertile plants. The methodscan also be used with transgenic animals.

2. Site Specific Gene Expression

Similar cell lines can also be used to produce proteins, metabolites orother compounds of biological or biotechnological interest using atransgene, a variety of promoters, regulators and/or structural genes.The gene will be always inserted at the same localisation in thechromosome. In transgenic animals, it makes possible to test the effectof multiple drugs, ligands, or medical proteins in a tissue-specificmanner.

3. Insertion of the I-Sce I recognition site in the CFTR locus usinghomologous sequences flanking the CFTR gene in the genomic DNA. TheI-Sce I site can be inserted by spontaneous gene replacement bydouble-crossing over (Le Mouellic et al. PNAS, 1990, Vol. 87,4712-4716).

4. Biomedical Applications

A. In gene therapy, cells from a patient can be infected with a I-Sce Icontaining retrovirus, screened for integration of the defectiveretrovirus and then co-transformed with the I-Sce I producing vector andthe donor sequence.

Examples of appropriate cells include hematopoeitic tissue, hepatocytes,skin cells, endothelial cells of blood vessels or any stem cells.

I-Sce I containing retroviruses include pG-MPL, pG-MtkPL or any kind ofretroviral vector containing at least one I-Sce I site.

I-Sce I producing vectors include pCMVI-Sce I(+) or any plasmid allowingtransient expression of I-Sce I endonuclease.

Donor sequences include (a) Genomic sequences containing the completeIL2 gene; (b) Genomic sequences containing the pre-ProInsulin gene; (c)A large fragment of vertebrate, including human, genomic sequencecontaining cis-acting elements for gene expression. Modified cells arethen reintroduced into the patient according to established protocolsfor gene therapy.

B. Insertion of a promoter (i.e., CMV) with the I-Sce I site, in a stemcell (i.e., lymphoid). A gap repair molecule containing a linker(multicloning site) can be inserted between the CMV promoter and thedownstream sequence. The insertion of a gene (i.e., IL-2 gene), presentin the donor plasmids, can be done efficiently by expression of theI-Sce I meganuclease (i.e., cotransfection with a I-Sce I meganucleaseexpression vector). The direct insertion of IL-2 gene under the CMVpromoter lead to the direct selection of a stem cell over-expressingIL-2.

For constructing transgenic cell lines, a retroviral infection is usedin presently available systems. Other method to introduce I-Sce I siteswithin genomes can be used, including micro-injection of DNA,Ca-Phosphate induced transfection, electroporation, lipofection,protoplast or cell fusion, and bacterial-cell conjugation.

Loss of heterozygosity is demonstrated as follows: The I-Sce I site isintroduced in a locus (with or without foreign sequences), creating aheterozygous insertion in the cell. In the absence of repair DNA, theinduced double-strand break will be extend by non-specific exonucleases,and the gap repaired by the intact sequence of the sister chromatide,thus the cell become homozygotic at this locus.

Specific examples of gene therapy include immunomodulation (i.e.changing range or expression of IL genes); replacement of defectivegenes; and excretion of proteins (i.e. expression of various secretoryprotein in organelles).

It is possible to activate a specific gene in vivo by I-Sce I inducedrecombination. The I-Sce I cleavage site is introduced between aduplication of a gene in tandem repeats, creating a loss of function.Expression of the endonuclease I-Sce I induces the cleavage between thetwo copies. The reparation by recombination is stimulated and results ina functional gene.

Site-Directed Genetic Macro-Rearrangements of Chromosomes in Cell Linesor in Organisms.

Specific translocation of chromosomes or deletion can be induced byI-Sce I cleavage. Locus insertion can be obtained by integration of oneat a specific location in the chromosome by “classical genereplacement.” The cleavage of recognition sequence by I-Sce Iendonuclease can be repaired by non-lethal translocations or by deletionfollowed by end-joining. A deletion of a fragment of chromosome couldalso be obtained by insertion of two or more I-Sce I sites in flankingregions of a locus (see FIG. 32). The cleavage can be repaired byrecombination and results in deletion of the complete region between thetwo sites (see FIG. 32).

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1. A method of inducing at least one site-directed double-strand breakin DNA of a cell, said method comprising (a) providing cells containingdouble-stranded DNA, wherein said DNA comprises at least one I-SceIrestriction site; (b) transfecting said cells with at least a plasmidcomprising DNA encoding the I-SceI meganuclease; and (c) selecting cellsin which at least one double-strand break has been induced. 2-22.(canceled)